JP2017081865A - Bone disease improver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bone disease improver useful for the prevention or treatment of bone disease.SOLUTION: A bone disease improver has as an active ingredient polysaccharides included in elephant breast milk and having a molecular weight of 80,000-600,000 dalton, the polysaccharides including as constituent sugar at least glucosamine and/or N-acetylglucosamine.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a bone disease ameliorating agent and a method for producing the same.

  Conventionally, breastfeeding of child elephants has been using artificial milk when breastfeeding is difficult due to the stress of the mother elephant and the lack of social education due to the inability to form a herd. As the artificial milk, those which are close to the general composition of elephant breast milk based on raw cow milk and dairy products, and vitamin and mineral blends whose composition is analogized based on the nutritional requirements of other animals and the like are used.

  However, until now, in elder milk, the elephant often fractures when the age is around 6 to 12 months, and countermeasures have been demanded (Non-patent Document 1).

  As for the breeding of Asian elephants (Indian elephants) in Japan, the female elephant “Z” bred at the Kobe City Oji Zoo gave birth to “M” in March 2004. October 2007 Gave birth to "O", but in both births, "Z" abandoned childcare and could not breastfeed, and two were raised with artificial milk. Then, “M” was 8 months old and “O” was 1 year and 1 month old, and fractures were confirmed (Non-Patent Documents 2 and 3).

  On the other hand, in the Ichihara country in Chiba Prefecture, female elephant “P” gave birth to “Y” in May 2007 and “R” in September 2013. Both “Y” and “R” are naturally nurturing (breastfeeding) with “P”, and the two have been steadily growing without being fractured by breastfeeding (Non-Patent Documents) 4).

  The “Z” at the Kobe City Oji Zoo subsequently moved to Ichihara Elephant Country and gave birth to “U” in June 2014, but also abandoned childcare there. Therefore, “P” will breastfeed “U” instead, and “U” has been growing smoothly without any fracture due to the mixed nutrition of “P” breast milk and artificial milk (non-patent) Reference 5).

As described above, it is considered that raising elephants using artificial milk has a higher risk of fractures than natural breastfeeding (breastfeeding). Causes of fractures include lack of sunshine and exercise, as well as differences in the composition and amount of nutrient intake between artificial milk and breast milk, but conventionally used artificial milk is related to bone growth. Nutrients to be taken into account were considered and there was no thought of any missing nutrients.
In Non-Patent Document 6, calcium, phosphorus and vitamin D are listed as important nutrients for forming bone.

By the way, glucosamine suppresses the decrease in bone density of ovariectomized mice (Non-patent Document 7), the effect of glucosamine hydrochloride on changes in subchondral bone in osteoarthritis model animals (Non-patent Document 8), and osteoarthritis of glucosamine The relationship between glucosamine and bone or cartilage has been reported, such as the effect on cartilage metabolism in (OA) (Non-patent Document 9). Furthermore, there is a report that oral arterial hyaluronic acid alleviates osteoarthritis pain (Non-patent Document 10).
Regarding polysaccharides containing glucosamine, it has been reported that a mixture of chondroitin sulfate and hyaluronic acid is used for treatment of joints exhibiting degeneration of articular cartilage (Patent Document 1).

In addition, there are some reports on oligosaccharides in elephant's breast milk (for example, Non-Patent Document 11), but there is no example of measuring the content of the constituent sugar, and the glucosamine content in the hydrolyzate is also known. There wasn't.

International Publication No. 2003/041724 Pamphlet

Emanuelson, K., Neonatal care and hand rearing.In: Fowler, M. E., and S. K. Mikota (Eds.), Biology, Medicine, and Surgery of Elephants.Blackwell Publishing, Ames, Iowa.p.233-241, 2006 "Peach (Elephant)", [online], [Search September 17, 2015], Internet <URL: https://en.wikipedia.org/wiki/%E3%83%A2%E3%83%A2_ (% E8% B1% A1)> "Ouji (elephant)", [online], [Searched on September 17, 2015], Internet <URL: https://en.wikipedia.org/wiki/%E3%82%AA%E3%82%A6 % E3% 82% B8 _ (% E8% B1% A1)> "Pulley", [online], [searched on September 17, 2015], Internet <URL: https://en.wikipedia.org/wiki/%E3%83%97%E3%83%BC % E3% 83% AA% E3% 83% BC _ (% E8% B1% A1)> "Ichihara Elephant Country", [online], [searched on September 17, 2015], Internet <URL: http://www.zounokuni.com/animals/> Hiroshi Morimoto “Revised Livestock Nutrition Correction 3rd Edition” Yokendo, November 10, 1971, p.338-339 Yamazaki, H. et al., Chitin and chitosan research 13 (2): 222, 2007 Wang S.X., Arthritis Rheum. 56 (5): 1537-1548, 2007 Jerosch, J., Int J Rheumatol.Volume 2011, Article ID 969012. Tashiro T. et al., The Scientific World Journal, Volume 2012, Article ID 167928 Uemura Y. et al., Comp. Biochem. Physiol. Part A Mol. Integr. Physiol., 145: 468-478, 2006

  An object of the present invention is to provide a bone disease ameliorating agent effective for the prevention or treatment of bone disease.

  In order to solve the above-mentioned problems, as a result of studies on mammalian breast milk, the inventors have found that a large amount of a specific polysaccharide is contained, and have completed the present invention.

That is, the present invention is a polysaccharide having a molecular weight of 80,000 to 600,000 daltons contained in mammalian breast milk, and contains at least glucosamine and / or N-acetylglucosamine as an active ingredient as an active ingredient, A bone disease ameliorating agent is provided.
The bone disease ameliorating agent has a preferred embodiment in which the mammal is an elephant.
In addition, the bone disease ameliorating agent is preferably configured such that the polysaccharide exhibits a molecular weight of about 270,000 daltons by high performance liquid chromatography.
In addition, the bone disease ameliorating agent is preferably used for oral intake.

  The present invention also provides a food or drink containing the bone disease-improving agent in an amount of 0.1% by mass or more in terms of solid matter as the content of the polysaccharide.

The present invention also provides a method for producing a bone disease-improving agent, wherein a polysaccharide having a molecular weight of 80,000 to 600,000 daltons is fractionated from elephant breast milk, and the obtained polysaccharide fraction is formulated as an oral composition. To do.
In the method, it is preferable that the mammal is an elephant.
Moreover, the said method makes it preferable to fractionate the polysaccharide which shows the molecular weight of about 270,000 daltons by a high performance liquid chromatography.

  The bone disease improving agent of the present invention is effective for prevention or treatment of bone disease.

High performance liquid chromatogram of elephant breast milk. High-performance liquid chromatogram of a hyaluronic acid sample (molecular weight of about 215,000 daltons). High-performance liquid chromatogram of artificial milk. Calibration curve of elution time in high performance liquid chromatography against the molecular weight of hyaluronic acid preparation (molecular weight 1.54 million to 7.45 million dalton). The vertical axis is log molecular weight, and the horizontal axis is elution time (minutes). MS / MS spectrum of a peak of retention time 8.6 min in LC-MS of a hydrolyzed sample of elephant milk.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.

The bone disease-improving agent of the present invention is a polysaccharide having a molecular weight of 80,000 to 600,000 daltons, preferably 120,000 to 540,000 daltons, more preferably 180,000 to 440,000 daltons contained in mammalian breast milk, A polysaccharide containing at least glucosamine and / or N-acetylglucosamine as a constituent sugar is contained as an active ingredient. The mammal includes animals having the characteristics of performing sexual reproduction and raising a child with milk.For example, platypus, opossum, cyloeopossum, wolf cat, bandicoot, owl mole, kangaroo, hedgehog, African shrew, Prickly pear, rock bream, elephant, dugong, armadillo, sloth, anteater, pineapple, lemur monkey, lemur, garago, monkey, gibbon, orangutan, gorilla, chimpanzee, bonobo, human, rabbit, rat, rat, mouse, guinea pig, squirrel, porcupine, Nutria, hedgehog, mole, camel, wild boar, giraffe, deer, cow, sheep, goat, hippo, whale, dolphin, pangolin, cat, raccoon dog, tiger, wolf, weasel, bear, seal, horse, rhinoceros, tapir, bat Etc. The. Among these mammals, elephants are preferred. Examples of elephants include Asian elephants and African elephants.
Hereinafter, such a polysaccharide may be referred to as “polysaccharide of the present invention” or simply “polysaccharide”. The polysaccharide preferably exhibits a molecular weight of about 270,000 daltons by high performance liquid chromatography.

The polysaccharide of the present invention is a polysaccharide having a molecular weight of 80,000 to 600,000 daltons, preferably 120,000 to 540,000 daltons, more preferably 180,000 to 440,000 daltons from the above-described mammals, for example, elephant breast milk. Can be obtained by fractionation. Molecular weight fractionation can be performed by gel filtration chromatography, high performance liquid chromatography, membrane separation using a microfiltration (MF filtration) membrane or ultrafiltration (UF filtration) membrane, and the like. Standards used for molecular weight measurement include hyaluronic acid, for example, hyaluronic acid having a molecular weight of about 215,000. The fact that the obtained fraction contains polysaccharides means, for example, the iodine starch method, the carbazole sulfate method, and the method using periodic acid and guanidine as a post-label reaction reagent (JP-A-8-6).
No. 2220) and the like. The polysaccharide of the present invention may be a purified product or a crude product, or may be a mixture with other components. The purity of the polysaccharide of the present invention in the polysaccharide fraction is not particularly limited, but is usually 0.1%, preferably 1%.

The polysaccharide of the present invention can be used as it is as a bone disease ameliorating agent (hereinafter also referred to as “the drug of the present invention”), or as an active ingredient of a food or drink or feed.
In one aspect, the drug of the present invention can be used as a medicine or an active ingredient thereof. The medicament of the present invention can be administered to mammals including humans as they are, or in combination with pharmaceutically acceptable pharmaceutical carriers. The administration route is preferably oral. In the present invention, it is preferable to exclude artificial milk for baby elephants.

  The preparation form of the drug of the present invention is not particularly limited, and tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules, soft capsules), granules (coated ones) ), Pills, troches, encapsulated liposomes, solutions, or pharmaceutically acceptable sustained-release preparations thereof. Addition of carriers, excipients, binders, disintegrating agents, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents, etc. Agents can be used. Moreover, unless the effect of this invention is impaired, you may use together polysaccharide and the chemical | medical agent or pharmaceutical composition which has the bone disease improvement effect | action known or discovered in the future. The pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or may be combined and commercialized as a separate drug without being contained in the drug of the present invention.

  Carriers and excipients used in the above formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders For example, starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like can be exemplified.

  Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.

  Furthermore, examples of lubricants include magnesium stearate, hydrogenated vegetable oil, and macrogol, and examples of colorants include red No. 2, yellow No. 4, and blue No. 1 that are allowed to be added to pharmaceuticals. can do.

  Tablets and granules include sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate, It can also be coated with a methacrylic acid polymer or the like.

One embodiment of the present invention is a polysaccharide having a molecular weight of 80,000 to 600,000 daltons, preferably 120,000 to 540,000 daltons, more preferably 180,000 to 440,000 daltons, or a high-speed liquid This is a method for producing a bone disease ameliorating agent, wherein a polysaccharide having a molecular weight of about 270,000 daltons is fractionated by chromatography, and the obtained polysaccharide fraction is formulated as an oral composition. Formulation can be performed in the same manner as described above. The polysaccharide fraction may be a purified or roughly purified polysaccharide. “Showing a molecular weight of about 270,000 daltons” means, for example, that the peak molecular weight of the fraction is about 270,000 daltons, for example 240,000 daltons to 300,000 daltons, preferably 40% or more of the peak area in the chromatogram Means that the molecular weight falls within the range of 180,000 to 440,000 daltons, with 60% or more, more preferably 75% or more.
Another aspect of the present invention is the use of the polysaccharide of the present invention in the manufacture of a medicament for improving bone diseases. Moreover, the other aspect of this invention is the said polysaccharide used for bone disease improvement. Another aspect of the present invention is a method for improving bone disease, wherein the polysaccharide is administered to a mammal.

  The amount of the polysaccharide contained in the drug of the present invention is not particularly limited and may be appropriately selected. % Or more, more preferably 50% by mass or more. The upper limit of the polysaccharide content is not particularly limited, and may be, for example, 90% by mass or less, 80% by mass or less, or 70% by mass or less.

  The agent of the present invention is useful for improving bone diseases. Improvement of bone disease includes prevention or treatment of bone disease. Bone diseases include fracture, refracture, bone defect, osteoarthropathy, osteoitis fibrosa, osteoporosis, osteomalacia ( osteomalacia), Paget's disease of bone, and rachitis. Furthermore, prevention of bone diseases includes reduction of the risk of fracture development, for example, increase in bone density, suppression of osteoclasts, and the like.

The administration time of the drug of the present invention is not particularly limited, and can be appropriately selected according to the state of the administration target.
The dose of the drug of the present invention is appropriately selected depending on the age, sex, condition, other conditions, etc. of the administration target. The amount of the polysaccharide of the present invention is preferably 0.01 to 100 mg / kg / day, preferably 0.1 to 50 mg / kg / day.
Further, when the administration period is long, for example, from 1 month to several months or more, the effect can be expected even when the dose of the drug is about 1/10 to 1/100 of the above range.
Regardless of the administration period, the drug can be administered once or divided into several times a day.

  The drug of the present invention or the polysaccharide which is an active ingredient thereof can be contained in a food or drink (beverage or food).

  Moreover, polysaccharides or the chemical | medical agent of this invention can be contained in food / beverage products as an active ingredient, and can be processed as food / beverage products which have a muscular-atrophy inhibitory effect | action as one aspect | mode of a bone disease improving agent.

  As the food and drink, the form and properties are not particularly limited as long as they can be taken orally without impairing the effect of improving bone disease, and usually using raw materials usually used for food and drink, except for containing polysaccharides. It can manufacture by the method of.

Foods such as the above, in the form of liquids, pastes, gels, powders, etc., in addition to tablet confectionery, liquid foods, etc., for example, bread, macaroni, spaghetti, noodles, cake mix, fried flour Wheat flour products such as bread crumbs; instant noodles, cup noodles, retort / cooked food, canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods Agricultural processed products such as canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, cereals (cereal processed products); canned marine products, fish ham and sausages, marine products, marine delicacies, Tsukudani Processed fishery products such as fish; canned and pasted livestock products; processed livestock products such as livestock ham and sausage; processed milk, milk drinks, yogurts, Milk and dairy products such as acid bacteria beverages, cheese, ice cream, formula milk powder, cream and other dairy products; fats and oils such as butter, margarines, vegetable oils; soy sauce, miso, sauces, processed tomato seasonings , Mirins, basic seasonings such as vinegar; cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings and foods; frozen foods, Frozen foods such as semi-cooked frozen foods and cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, jelly, other confectionery Sweets such as carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruit granules, vegetable drinks, soy milk, soy milk Beverages, coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, other beverages such as other beverages, baby food, sprinkles, other marketed foods such as tea crush paste, etc .; Examples include infant formulas; enteral nutrition; functional foods (special health foods, nutritional functional foods) and the like.

  Furthermore, the polysaccharide or the agent of the present invention can be contained in the feed as an active ingredient, and can be processed as a feed having a bone disease improving action as one embodiment of the bone disease improving agent.

  The form of the feed is not particularly limited, for example, grains such as corn, wheat, barley, rye, milo; vegetable oils such as soybean oil meal, rapeseed oil meal, coconut oil meal, linseed oil meal; bran, wheat straw, rice bran, Dehydrated rice bran, etc .; Manufactured deer such as corn gluten meal, corn jam meal; Animal feed such as fish meal, non-fat dry milk, whey, yellow grease, tallow; Yeasts such as torula yeast, beer yeast; It can be produced by blending mineral feed such as calcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.

  The amount of the polysaccharide of the present invention contained in the food and drink (including feed) of the present invention is not particularly limited and may be appropriately selected, but is preferably 0.1% by mass or more, more preferably 1% by mass or more. It is good to do. The upper limit of the content of the polysaccharide of the present invention is not particularly limited, but may be, for example, 90% by mass or less, 80% by mass or less, or 70% by mass or less.

  Moreover, the food / beverage products (including feed) of the present invention contain 0.01 mg or more, preferably 0.1 mg or more, more preferably 1 mg or more of the polysaccharide of the present invention in terms of solids per single intake. desirable.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
Artificial milk (75.2 g of Wakodo “Yesi” + 6 g of palm oil + 200 g of hot water) used for nursing the Asian elephant “M” bred at the Kobe City Oji Zoo and the Asian elephant “Z” bred at the zoo (2014) General nutritional components (protein, lipids, minerals, vitamins) in breast milk of Asian elephant “P” (born September 3, 2013) bred in Chiba Prefecture Ichihara Elephant Country Was analyzed according to the official law (Japan Food Sanitation Association “Food Sanitation Inspection Guidelines RIKEN 2015”). The results are shown in Table 1. Hiromoto Morimoto, “Revised Livestock Nutrition Correction, Third Edition,” Yokendo, November 10, 1971, p.338-339, mentions calcium, phosphorus, and vitamin D as important nutrients that form bone. However, in the above artificial milk, all nutrients contained amounts above the average value of breast milk measurement.

Next, polysaccharide analysis of elephant breast milk (“Z”) and artificial milk was performed. Elephant breast milk and artificial milk were each centrifuged (12000 g, 10 minutes) to remove the fat fraction, and the resulting skim milk filtered through a 0.2 μm filter was used as an analysis sample. After separating the components in the sample by high performance liquid chromatography according to the molecular weight, polysaccharides were selectively detected by the post-column method (separation columns: “TSKgel G5000 PWXL”, “TSKgel G6000 PWXL”) Co., Ltd.), “PLRP-S 1000Å” (Agilent Technology Co., Ltd.) 3 linked, column temperature: 40 ° C., mobile phase: 0.195M sodium chloride / 2% acetonitrile mixture, mobile phase flow rate: 0. 8 ml / min, reaction solution: 45 mM guanidine hydrochloride / 4.5 mM sodium periodate / 150 mM boric acid mixture (pH 9.5), reaction solution flow rate: 0.4 ml / min, reaction temperature: 170 ° C. As a molecular weight standard, hyaluronic acid (R & D System) having a molecular weight of about 215,000 daltons was used.
The analysis results are shown in FIGS. In this system, which elutes faster as the molecular weight increases, a peak of high molecular polysaccharide (23.5 minutes) was detected from the elephant breast milk sample (FIG. 1). The molecular weight of this peak was estimated to be approximately 270,000 daltons from a calibration curve (FIG. 4) of elution time with respect to molecular weight, which was prepared using a hyaluronic acid sample having a molecular weight of 1.54 million to 7.45 million daltons. The rise and fall of this peak were a molecular weight of about 80,000 daltons and about 600,000 daltons, respectively, and the half width was about 180,000 to about 440,000 daltons. On the other hand, the peak was not detected from the artificial milk.
The content of polysaccharides with a peak molecular weight of 270,000 daltons in elephant breast milk was calculated to be 0.045% by mass in terms of solid matter.

Next, a sample having a molecular weight of 270,000 Dalton detected from elephant breast milk was collected and hydrolyzed with hydrochloric acid (6N HCl, 110 ° C., 3 hours) (hereinafter referred to as “hydrolyzed sample”). Analyzed by liquid chromatography mass spectrometry (LC-MS) (column: Intrada Amino Acid (3 μm) 2.0 mm ID × 150 mm L., column temperature: 40 ° C., mobile phase A: 0.2% formic acid / acetonitrile, mobile phase B : 100 mM ammonium acetate aqueous solution, flow rate: 0.2 ml / min, gradient: A / B: 50/50 → (10 minutes) → 0/100 → (5 minutes) → 0/100 → (1 minute) → 50/50 → (minutes). In addition, a glucosamine solution (added with 100 mM sodium chloride) was also analyzed.

As a result, in the analysis of the hydrolyzed sample, a peak was detected at the same retention time (8.6 minutes) as in the analysis of glucosamine (100 mM salt added), and the MS / MS spectra of the peaks were also consistent (FIG. 5).
Therefore, it became clear that the hydrolysis sample contained glucosamine. The high molecular weight polysaccharide having a molecular weight of 270,000 daltons derived from elephant breast milk is considered to contain at least glucosamine or N-acetylglucosamine as a constituent sugar.

Claims (8)

  A bone disease-improving agent comprising a polysaccharide having a molecular weight of 80,000 to 600,000 daltons contained in mammalian breast milk and comprising at least glucosamine and / or N-acetylglucosamine as a constituent sugar as an active ingredient.   The bone disease-improving agent according to claim 1, wherein the mammal is an elephant.   The bone disease-improving agent according to claim 1 or 2, wherein the polysaccharide exhibits a molecular weight of about 270,000 daltons by high performance liquid chromatography.   The bone disease-improving agent according to any one of claims 1 to 3, which is for oral intake.   Food / beverage products which contain the bone disease improving agent as described in any one of Claims 1-4 as 0.1 mass% or more in conversion of solid substance as content of the said polysaccharide.   A method for producing a bone disease-improving agent, wherein a polysaccharide having a molecular weight of 80,000 to 600,000 daltons is fractionated from mammalian breast milk, and the obtained polysaccharide fraction is formulated as an oral composition.   The method of claim 6, wherein the mammal is an elephant.   The method according to claim 6 or 7, wherein a polysaccharide having a molecular weight of about 270,000 daltons is fractionated by high performance liquid chromatography.