CN107006681A - Animal feed composition containing Chinese medicinal material extract - Google Patents
Animal feed composition containing Chinese medicinal material extract Download PDFInfo
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- CN107006681A CN107006681A CN201710045587.1A CN201710045587A CN107006681A CN 107006681 A CN107006681 A CN 107006681A CN 201710045587 A CN201710045587 A CN 201710045587A CN 107006681 A CN107006681 A CN 107006681A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/65—Amphibians, e.g. toads, frogs, salamanders or newts
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/232—Angelica
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/234—Cnidium (snowparsley)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/237—Notopterygium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/286—Carthamus (distaff thistle)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/39—Convolvulaceae (Morning-glory family), e.g. bindweed
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/43—Cuscutaceae (Dodder family), e.g. Cuscuta epithymum or greater dodder
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/486—Millettia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
- A61K36/638—Ligustrum, e.g. Chinese privet
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/80—Scrophulariaceae (Figwort family)
- A61K36/804—Rehmannia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9064—Amomum, e.g. round cardamom
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Abstract
The present invention relates to a kind of animal feed composition containing Chinese medicinal material extract.The animal feed composition containing Chinese medicinal material extract of one embodiment of the invention can will be used as active ingredient including the Chinese medicinal material extract containing dry Rana esculenta, the fruit of glossy privet and prepared rhizome of rehmannia.The animal feed composition of the present invention has extraordinary effect to preventing and treating the bone related diseases such as osteoporosis, arthritis, fracture, malacosteon aspect.
Description
Technical field
The present invention relates to animal feed composition, more particularly to a kind of animal feed composition containing Chinese medicinal material extract.
Background technology
Vegetables that current era, the mankind or animal eat or feed are due to microelement deficiencies and ring caused by the disaster of continuous cropping
Border pollution causes disease to be on the increase, therefore is generally directly or indirectly excessively taking the medicines such as biocide, and biocide is produced
The virus of raw drug resistance and bacterium cause more harm to the mankind and animal.
Through showing, particularly domestic animal lacks trace element and dysgenesia occurs and can not normally breed, micro when lacking
The health of the mankind or animal is harmful in the case of element.Therefore, various factors exploitation animal feed supply domestic animal is combined at present.
Relevant technical literature is referring to Korean granted patent the 10-0863607th.
In addition, with current aging, the treating bone diseases such as osteoporosis, fracture, arthritis are in steeply rising trend.It is raw
Body reaches balance to keep homeostasis always by the bon e formation of osteoblast and the bone information of osteoclast.Osteoporosis
It is that, because osteoblast is unbalance with osteoclast, the bone information of osteoclast occupies advantage than the bon e formation of osteoblast and occurred
Disease, what is be currently known has female hormone caused by the women hypofunction of ovary such as amenorrhoea to lack the reason for causing osteoporosis
Weary, aging and lack of exercise, the side effect of steroids medicine series etc..Therefore, in order to effectively treat or prevent osteoporosis,
Need to increase the activity of osteoblast or suppress to induce the activity of the osteoclast of bone information.
Accordingly, it would be desirable to study the material of the nutrient containing the mankind or animal shortage or can prevent may in the mankind or
The material for the disease that animal occurs.
The content of the invention
Technical problem
Extracted it is an object of the invention to provide a kind of Chinese medicine that contains that can effectively prevent or treat bone related disease
The animal feed composition of thing.
Technical scheme
To reach above-mentioned purpose, one embodiment of the present of invention, which discloses one kind, will contain dry Rana esculenta, the fruit of glossy privet and cultivated land
Yellow Chinese medicinal material extract as active ingredient animal feed composition.
Technique effect
It is according to an embodiment of the invention using Chinese medicinal material extract as active ingredient animal feed composition to pre-
There is extraordinary effect in terms of the bone related diseases such as anti-and treatment osteoporosis, arthritis, fracture, malacosteon.
Also, animal feed composition according to an embodiment of the invention suppresses to be responsible for point of the osteoclast of bone information
Change, therefore there is extraordinary effect to preventing and treating the treating bone diseases such as osteoporosis, animal feed can be used as
Additive.
Brief description of the drawings
Fig. 1 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of effect osteoclast differentiation in terms of is induced osteoclast differentiation factor (RANKL)；
Fig. 2 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of osteoclast generation is suppressed；
Fig. 3 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the bone information of osteoclast is suppressed；
Fig. 4 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the TRAP performances of osteoclast is suppressed；
Fig. 5 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the MMP-9 performances of osteoclast is suppressed；
Fig. 6 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of cathepsin K (Cathepsin K) performance of osteoclast is suppressed；
Fig. 7 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the NFATc1 performances of osteoclast is suppressed；
Fig. 8 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the c-fos performances of osteoclast is suppressed；
Fig. 9 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of cytotoxicity result.
Embodiment
Illustrate related to one embodiment of the invention animal feed composition and its system containing Chinese medicinal material extract below
Make method.
The odd number form of expression as used in this specification specification without other clearly state in the case of also include it is multiple
The number form of expression.In this manual, the term such as " composition " or " comprising " should not be construed as to include remembering in all specifications
Each inscape or each step carried, but be interpreted as which part inscape or part steps, or reason can not be included
Solve that other inscapes or step can also be included.
Contained Chinese medicinal material extract can be extracted by a variety of methods in the animal feed composition of one embodiment of the invention
Obtain.
Specifically, the Chinese medicinal material extract of the Multiple components including the frog can by cold water extraction, hot water extract, it is organic
The method such as solvent extraction or distillation is extracted and obtained.On the extract solution of extraction, directly it can be extracted using the Chinese medicine
Thing, can also be concentrated under reduced pressure or the extract solution of freeze-dried extraction prepares Chinese medicinal material extract.
Extracted for example, obtaining Chinese medicine by the solvent extraction of the group constituted selected from water, rudimentary alcohol and its mixed solution
Thing.The Chinese medicinal material extract of one embodiment of the invention can not only represent that therefrom medicinal material extract is obtained using water or specific solvent
Material in itself, also may indicate that by strain fermentation extract obtain the material or further thin with organic solvents such as ether
Divide extraction or the extraction machining object obtained by working processes such as post (column) divisions etc..
The Chinese medicine can contain following composition.
The Chinese medicine can contain dry Rana esculenta.The Rana esculenta is sweet cool in nature.Also, the Rana esculenta, which has, goes pyrolysis
The effect of poison, qi-restoratives, tonifying Qi, inducing diuresis to remove edema.Also, the Rana esculenta can eliminate the fire caused by fatigue, physically-draining, solve
Part is firmly condensed in blood, eliminates scorching in blood, to slow-growing animal (for example：Pig) effect is notable.Also, it is described edible
It is notable that the frog treats also effect to acute bloating, carbuncle and private parts furuncle.
The Chinese medicine can also include at least one of the fruit of glossy privet, reticulate millettia and kardiseed, be used relative to the xerophagia
The parts by weight of the frog 1, its ratio is respectively 1.5 to 3.5 parts by weight.
The Chinese medicine can also include prepared rhizome of rehmannia, the Radix Astragali, the bighead atractylodes rhizome, Du Chong, cortex acanthopanacis, the root of bidentate achyranthes, bark of ash, scythian lamb rhizome,
At least one of psoralea corylifolia, the rhizome of davallia, teasel root, cynomorium songaricum, thizoma curculiginis and maltose, relative to the dry parts by weight of Rana esculenta 1, its ratio
Respectively 0.5 to 1.5 parts by weight.
The Chinese medicine can also include at least one of levisticum and notopterygium root, relative to the dry parts by weight of Rana esculenta 1, its
Ratio is respectively 0.2 to 1.5 parts by weight.
The Chinese medicine can also include at least one of Semen Cuscutae, frutus cnidii, tribute fructus amomi, cassia twig and radix glycyrrhizae, relative to
The parts by weight of dry Rana esculenta 1, its ratio is respectively 0.1 to 1.3 parts by weight.
Specifically it can be prepared as follows：
It is possible, firstly, to screen in the above-mentioned a variety of Chinese medicine composition including including Rana esculenta at least a portion and be made appropriate big
It is small., can be on the basis of weight with C1 to C4 such as water, ethanol, the methanol of about 1 to 50 times of component for the Chinese medicine being made
Rudimentary alcohol or its mixed solvent by carrying out the stirring extraction of about 1 to 100 hour at such as 5 DEG C to 100 DEG C, hot soup carries
Take, cold soaking is extracted, the extracting method such as refluxing extraction or ultrasonic wave extraction filtered to the extract solution of acquisition, concentrated or dry
To Chinese medicinal material extract.The extract can be carried out with the organic solvent such as water or rudimentary alcohol, ether if necessary
Further extract and use.
The Chinese medicinal material extract of one embodiment of the invention can effectively hinder the differentiation of osteoclast.Also, the present invention
The Chinese medicinal material extract of one embodiment suppresses to be responsible for the TRAP, MMP-9, cathepsin K (Cathepsin K) of bone information
Show, the genetic performance such as NFATc1, MITF occurred when can suppress the differentiation of osteoclast with activity increase.
In addition, the Chinese medicinal material extract of one embodiment of the invention can regard Lignum Aquilariae Resinatum extract as active ingredient by manufacturing
Pharmaceutical composition or functional food general formulation method be made.Herein, Lignum Aquilariae Resinatum extract is removed in Chinese medicinal material extract
Outside can also be used as needed containing a variety of auxiliary raw medicinal herbs, vitamin, mineral matter, dietary fiber and other are medical-food
Adjuvant (for example, excipient, reinforcing agent, coating agent etc.) etc..
Osteoclast (osteoclast) is the monokaryon derived cell of hemocytoblast, itself and Gegenbaur's cell (osteo
Blast it is) common that bone uptake, aggregate formation and healing fracture are played an important role.Osteoclast differentiation needed for the factor have core because
Sub- kB receptor activations factor ligand (receptor activator of nuclear factor kapp a B ligand；
RANKL), tumor necrosis factor α (tumor necrosis factor α；TNFα)、N F-kB、ODF(osteoclast
Differentiation factor) etc..
Especially, RANKL is that osteoclast breaks up the essential factor.The RAW264.7 monokaryon glomus cells of mouse pass through
RANKL (receptor activator of nuclear factor kappa B ligand) is merged and is divided into multinuclear and breaks
Osteocyte (multinucleated osteoclast).RANKL secrete in activation osteoblast or stem cell or into
Fibrocyte and with RANK (the receptor activator of positioned at osteoclastic precursor and osteoclast surface
Nuclear factor kappa B) combine the activity for promoting MAPK, be called NF-kB transcription factor enter in core increase with it is broken
The related gene of bone cell differentiation is TRAP (tartrate-resistant acidphosphatase), MMP-9
(metalloproteinase-9), the performance of cathepsin K (Cathepsin K) etc. to absorb bone.Also, differentiation
Osteoclast is transmitted by TRAF6 (TNF associated factor 6) and c-Fos signals causes NFATc1 (nuclear f
Actor of activated T cell 1) etc transcription factor activity.
Osteoclastic precursor can be passed through by these atomization formation osteoclasts even, the osteoclast of formation
The effect of TRAP, MMP, cathepsin K (Cathepsin K) etc. absorbs bone induced osteoporosis disease.
The animal feed composition of one embodiment of the invention is containing Chinese medicinal material extract etc., therefore, it is possible to effectively suppress osteoclastic
Cell differentiation, prevention or treatment for osteoporosis.
The animal feed composition can pass through medicine well known in the art using Chinese medicinal material extract as its active ingredient
The carrier that the addition of product preparation method pharmaceutically allows.
The usable carrier for example can be lactose, sucrose, sorbierite, mannitol, xylitol, antierythrite, malt
Sugar alcohol, starch, acacia glue (acacia gum), alginic acid glue, gelatin, calcium phosphate, calcium silicates, cellulose, methylcellulose, crystallite
Cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, Nipasol, talcum powder, magnesium stearate and mineral oil etc., but the present invention
Not limited to this.
Also, the lipase inhibitor can also include filler, anti-coagulants, lubricant, wetting agent, spices, emulsification
Agent or preservative etc..The animal feed composition of one embodiment of the invention can be melted into according to usual way formulation respectively to be dissipated
The oral type such as agent, granule, lozenge, capsule, suspension, emulsion, syrup, aerosol formulation, external preparation, suppository and sterilizing note
The form for penetrating solution is used.
The animal feed composition containing Chinese medicinal material extract of the present invention can also be dredged to prevent or treat sclerotin
Loose disease, osteoarthritis and Clinical practice.On input amount, input number of times and the throwing of the Chinese medicinal material extract put into patient
Angle of incidence etc., can optionally add and subtract regulation according to conditions such as the sex of patient, age, state, body weight, environment.But, one
As can with input on the one three times and it is specific put between each meal, can be by 0.1mg to 2000mg amount point one to putting into for several times.
The functional food of one embodiment of the invention is containing Chinese medicinal material extract etc., therefore, it is possible in osteoporosis, bone
Improvement is played in terms of arthritis.Signified food not only includes directly absorbing the food of state in this specification, also
Include the broad concept of food materials, food additives, food material etc..The functional food individually can directly add it is described in
Medicinal substances extract is used, but it is also possible to is added known substance or novel substance for preventing, treating osteoporosis and is helped
In bone health known material or novel substance with humidification.And it is possible to use food coloring, flavorant, crude drug
The general additive such as grass, fruit, oligosaccharide, ketone acid, citric acid causes taste and pleasant aroma.
Functional food containing the Chinese medicinal material extract may include Biscuits, ice-cream products class, breast processing category, meat
Product class, fish product class, noodles, beverage, seasoned food, margarine, shortening, Pizza etc..
The Biscuits includes bread, cake class, dry fruit, carbohydrate, chocolate-like, chewing gum, jam etc., the ice river in Henan Province
Drenching product class may include ice cream, ice fruit, ice cream powdery type etc..
It is described breast processing category may include cow's milk class, low fat cow's milk class, lactose decompose cow's milk, modified milk class, Goat Milk,
Acidified milk class, skimmed milk class, yoghurt class, samneh class, skimmed milk, natural cheeses, processed cheese, milk powder class, whey class
Deng.
The meat products class may include meat processed goods, egg processed goods, hamburger etc., fish product class may include fish cake, ham,
Sausage, bacon etc..
The noodles may include hand-pulled noodles class, dried noodle class, noodle, instant fried flour class, luxurious dried noodle class, improve ripe noodles,
Cold winter noodles, Italian noodles etc., the beverage may include fruit beverage, greengrocery beverage, soda, soya-bean milk class, Yoghourt
Deng sour milk beverage, bland etc..Also, the seasoned food may include soy sauce, salty sauce, thick chilli sauce, sweet fermented flour sauce, clear state
Sauce, mixing sauce, vinegar, sauce class, catsup, curry, flavouring etc..
Also, the animal feed composition containing Chinese medicinal material extract related to one embodiment of the invention can be made into curable type
Ball, liquid-state feed, semi solid slurry form.
The detailed description below experimental example related to one embodiment of the invention.Following experimental example is to understand this hair for help
It is bright and provide, the scope of the present invention is not restricted by the embodiments.Following experimental example will represent that Chinese medicine is extracted with DBT
Thing.
1. the preparation of Chinese medicinal material extract (DBT)
Chinese medicine containing dry Rana esculenta is pulverized into flow back in triplicate 30% ethanol 5L, time-consuming three hours after fritter and carried
After extract operation, concentrated extracting solution obtains Chinese medicinal material extract (DBT).Pick-up rate is 17.3%.
2. osteoclast Analytical Chemical Experiment
1) RAW264.7 cells are cultivated
Utilize DMEM (Dulbecco's modified eagle medium)/10%FBS (fetal bovine
Serum)/PC-SM trainings ground is in CO2 cell culture medium culture RAW264.7 cells, and cell number is 5x103 cells/wells
(5x103cells/well), is cultivated using 96 orifice plates (96well plate).Culture abandons nutrient solution and changed after 24 hours
Cell is cultivated into the a-MEM added with 10%FBS, 50ng/ml RANKL, 1ng/ml TGFb.It with the addition of to nutrient solution various dense
The Chinese medicinal material extract (DBT) of degree.Cultivated with changing within every two days an identical training.
2) osteoclast differentiation and generation are determined
With RANKL by after the cell inductions of RAW 264.7 to osteoclast, dyeing is acknowledged as the performance of mature osteoclast
Indicate tartrate-resistant acid phosphatase (the tartarate-resistance aci d phosphatase of (marker)；
TRAP TRAP-positive cells) are confirmed.The cell broken up twice with PBS, with 3.7% formaldehyde-citrate-the third
Ketone (formaldehyde-citrate-acetone) solution is fixed 10 minutes and cleaned twice with distilled water.With including by identical
Ratio mixes the quick garnets of 2%TRAP (fast garnet) GBC aqueous slkalis and NaNO3Solution and 5% stone that solution is obtained
Cerebrol AS-BI phosphoric acid (naphtha AS-BI phosphoric acid), 4% acetic acid (acetic acid), 2% tartaric acid
The solution of (tartaric acid) is handled fixed cell and in room temperature more than 30 minutes.Use light microscope
Observed and count positive (TRAP-positive) apocytes (TRAP (+) MNCs) of TRAP that core is more than three and counted
And generate index as osteoclast.
Fig. 1 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect in terms of osteoclast differentiation factor (RANKL) induces osteoclast differentiation.
As illustrated, promoting osteoclast differentiation, the Chinese medicine of one embodiment of the invention in the case of being handled with RANKL
Material extract (DBT) suppresses TRAP (+) MNCs formation to significantly inhibit osteoclast differentiation.It can thus be appreciated that the present invention one
The Chinese medicinal material extract (DBT) of embodiment has the effect by suppressing the differentiation blocking bone information of osteoclast.
3) measure of osteoclast function
On the activity of osteoclast, whether suppressed by determining TRAP determinations of activity.Determining TRA P activity aspects makes
With the tool set (Sigma-387A) bought from Sigma companies.TRAP activity is scaled absorbance for control group
Ratio represent, intentional property (* p are demonstrated by t-test<0.05, * * p, 0.01, * * * p, 0.001).
Fig. 2 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of osteoclast generation is suppressed.
As illustrated, Fig. 2 suppresses to participate in the anti-tartaric acid of the bone information of osteoclast for display Chinese medicinal material extract (DBT)
Hydrochloric acid acid phosphatase (tartarate-resistance acid phosphatase；TRAP the coordinate diagram of active effect).
It can thus be appreciated that the Chinese medicinal material extract (DBT) of one embodiment of the invention effectively suppresses osteoclastic by significantly inhibiting TRAP activity
The activity of cell.
4) measure of osteoclast gene performance
(1) total serum IgE is separated
After removing culture cell training ground for separation RNA (washi ng) three is cleaned with phosphate buffer (4PBS)
It is secondary.Collect after cell in 750 × g centrifugations 10 minutes, then cleaned twice with ice-cold PBS (icec old PBS).Xiang Chen
Centrifuged after cell addition lysis buffer (lysis buffer) dissolving in shallow lake in 12,000 × g and take upper strata in 10 minutes.
In 36,000rpm after being placed on again on sucrose containing 1M (sucrose) TRIS buffer (Tris buffer)
Centrifuge 160 minutes.Polysome ball (Polysome pellet) is taken to be dissolved in dodecyl sodium sulfate buffer solution (SDS
Buffer (incubation) is hatched 30 minutes at 37 DEG C after).Add after sodium-acetate buffer (Sod.Acetate buffe r)
Extracted with phenol/CsKl3 solution.Add after NaCl to 0.2M addition EtOH and in -20 DEG C of keepings 12 hours.It is clear with 75%EtOH
It is dried after washing sediment twice.RNA amounts needed for adjustment gene performance cause control group and mRNA amounts phase in trial flock
Confirmed and used with electrophoresis with after.
(2) cDNA preparation
To the μ l (RNA containing 10ug) of the total serum IgE liquid 10 addition oligosaccharide (oligo) separated respectively from control group and trial flock
Carefully mixed after the μ l of dT 1 (2ug/ μ l), then hatch (incubation) five minutes at 90 DEG C.About 10 points are placed in room temperature
Clock to be maintained at 4 DEG C after primer (primer) annealing (annealing) and adds reagent.Add 5x Sai Keli buffer solutions
(cyscript buffer) 4 μ l, 0.1M dithiothreitol (DTT) (DTT) 2 μ l, dUTP mixed nucleotides (nucleotide mix) 1 μ
The μ l, Sai Keli reverse transcriptases (Cyscr of l, dUTP Cy dye labeled nucleotides (Cydye-labelled nucleotide) 0.1
Ipt reverse transcriptase) 1 μ l, H2O 0.9 μ l carefully to be tilted with hand after reaching 20 μ l
(tipping) mix.It is put on ice for after hatching (incubation) 90 minutes at 42 DEG C afterwards.Herein, it is each to add 2 μ l's
Hatch (incubation) 15 minutes at 37 DEG C after 2.5M NaOH, add 10 μ l 2M hydroxyethyl piperazine second sulphur acid buffers
(HEPES buffer) (free acid are free acid) is neutralized.Each reagent is purchased from Amersham Bioscience.
(3) realtime quantitative inspection (Real Time Polymerase Chain Reaction) (real-time quantitative
PCR；real time-PCR)
To each optical tube (optical tube) (Optical 96-Well Reaction Plate
With Barcode and Optical Adhesive Films, Applied Biosystems, Cat.No.4314320) add
Plus the μ l (Sigma-Aldrich, Cat.No.S9430) of SybrGreen Mix 2.5 of three times, the μ l of cDNA 1 of above-mentioned synthesis,
10pmol/ μ l pair of primers combination (primer pair mix) 1 μ l, the buffering of 2.5m M μ l, the 10xTag polymerases of dNTP 2
(polymerase buffer) 2.5 μ l, the μ l of label aggregation enzyme (Tag Polymerase) 0.3 and 14.7 μ l H2O, and be amplified to
95 DEG C of 5 minutes 1 cycles, 95 DEG C 30 seconds, 45 DEG C 30 seconds, 72 DEG C of 60 seconds 40 cycles, 95 DEG C of 20 minutes 1 cycles.Primer (P rimer)
It make use of the material used in reverse transcriptase-Polymerase Chain Reaction (RT-PCR).Terminate polymerase chain reaction (PCR) to take out afterwards
Pipe, then determines polymerase chain reaction specificity (PCR with the μ l of reaction solution 5 in 3% Ago-Gel (agarose gel)
Specificity), use7000 sequence detection systems (Sequence Detection System)
(AppliedBiosystems, Cat.No.4349157) analyzes real-time quantitative PCR (real time PCR) result.
Primer for PCR is as shown in table 1 below.
【Table 1】
Fig. 3 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the bone information of osteoclast is suppressed.
Coordinate diagram shown in Fig. 3 be Chinese medicinal material extract (DBT) to participate in osteoclast osteoclasia TRAP it is active
Inhibition.
Fig. 4 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the TRAP performances of osteoclast is suppressed.
Coordinate diagram understands to show inhibition with Chinese medicinal material extract (DBT) to the TRAP of osteoclast as shown in Figure 4
Amount increase and increase.
Fig. 5 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the MMP-9 performances of osteoclast is suppressed.
Inhibition is showed with the amount increase of Chinese medicinal material extract (DBT) to the MMP-9 of osteoclast as shown in Figure 5
Increase.
Fig. 6 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of cathepsin K (Cathepsin K) performance of osteoclast is suppressed.
As shown in Figure 6 coordinate diagram understand to show the cathepsin K (Cathepsin K) of osteoclast inhibition with
The amount increase of Chinese medicinal material extract (DBT) and increase.
Fig. 7 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the NFATc1 performances of osteoclast is suppressed.
Coordinate diagram understands to show inhibition with Chinese medicinal material extract to the NFATc1 of osteoclast as shown in Figure 7
(DBT) amount increases and increased.
Fig. 8 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of the effect of the c-fos performances of osteoclast is suppressed.
Coordinate diagram understands to show inhibition with Chinese medicinal material extract (DBT) to the c-fos of osteoclast as shown in Figure 8
Amount increase and increase.
Fig. 9 Chinese medicinal material extracts contained by showing in the animal feed composition related to one embodiment of the invention
(DBT) schematic diagram of cytotoxicity result.
Coordinate diagram understands that the toxicity of cell does not increase and increased with the amount of Chinese medicinal material extract (DBT) as shown in Figure 9.
As described above, contained Chinese medicine composition to the good crude drug of safety, effect from adding in animal feed composition
Plus the Chinese medicine of Rana esculenta is constituted, the treatment and preventive effect to bone metabolic disease are notable.
As described above, one embodiment of the invention using Chinese medicinal material extract as active ingredient animal feed composition
Prevention or treatment to bone related diseases such as osteoporosis, arthritis, fracture, malacosteons have extraordinary effect.
Also, animal feed composition according to an embodiment of the invention suppresses to be responsible for point of the osteoclast of bone information
Change, therefore there is extraordinary effect to preventing and treating the treating bone diseases such as osteoporosis, animal feed can be used as
Additive.
Animal feed composition described above containing Chinese medicinal material extract and preparation method thereof is not limited to described above
The composition and method of embodiment, can be by all embodiments or optionally combination a portion embodiment is implemented to described
Example carries out various deformation.
Claims (5)
1. a kind of animal feed composition, it is characterised in that contained active ingredient is to include dry Rana esculenta, the fruit of glossy privet and prepared rhizome of rehmannia
Chinese medicinal material extract.
2. animal feed composition according to claim 1, it is characterised in that：
The Chinese medicinal material extract also includes at least one of reticulate millettia and kardiseed.
3. animal feed composition according to claim 1, it is characterised in that：
The Chinese medicinal material extract also includes at least one of levisticum and notopterygium root.
4. animal feed composition according to claim 1, it is characterised in that：
The Chinese medicinal material extract also includes at least one of Semen Cuscutae, frutus cnidii, tribute fructus amomi and cassia twig.
5. animal feed composition according to claim 1, it is characterised in that：
Ratio relative to the fruit of glossy privet contained by the dry parts by weight of Rana esculenta 1 described in the Chinese medicinal material extract is 1.5 to 3.5
Parts by weight, the ratio of the contained prepared rhizome of rehmannia is 0.5 to 1.5 parts by weight.
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| WO2022237873A1 (en) * | 2021-05-14 | 2022-11-17 | 天津中医药大学 | Pharmaceutical composition for preventing and treating osteoporosis |
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| KR20180095313A (en) | 2017-02-17 | 2018-08-27 | 최하영 | Herbal composition and the manufacturing method for pat improving circulation of blood |
| KR20190014386A (en) | 2017-08-02 | 2019-02-12 | 주식회사 아미텍 | Poultry feed additive for immunity enhancement using korea herbal medicine byproduct and fermented red ginseng red ginseng residue |
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