CN108404107A - A kind of compound preparation and preparation method thereof of the tree peony peptide with strengthen immunity - Google Patents
A kind of compound preparation and preparation method thereof of the tree peony peptide with strengthen immunity Download PDFInfo
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- CN108404107A CN108404107A CN201810282458.9A CN201810282458A CN108404107A CN 108404107 A CN108404107 A CN 108404107A CN 201810282458 A CN201810282458 A CN 201810282458A CN 108404107 A CN108404107 A CN 108404107A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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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
- A61K36/481—Astragalus (milkvetch)
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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)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8969—Polygonatum (Solomon's seal)
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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)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Abstract
The invention discloses a kind of compound preparations of the strengthen immunity of peptide containing tree peony, are prepared by the raw material of following parts by weight：36 parts of dendrobium candidum solid powder, 48 parts of Radix Astragali solid powder, 24 parts of sealwort solid powder, 6 10 parts of tree peony peptide.Dendrobium candidum solid powder is dendrobium candidum water extract or dendrobium candidum extractive with organic solvent；Radix Astragali solid powder is Radix Astragali water extract or Radix Astragali extractive with organic solvent；The sealwort solid powder is sealwort water extract or sealwort extractive with organic solvent；Tree peony peptide solid powder is extract of the tree peony albumen after complex enzyme hydrolysis.The preparation of the present invention has effects that strengthen immunity.It can not only promote proliferation transformation, the mononuclear macrophage carbonic clearance ability of mouse spleen lymphocyte, while also improve the ability of antibody-producting cell, promote the delayed allergy of immunodeficiency models mouse caused by anti-hydrocortisone.In addition, being had not significant impact to the ratio of weight, internal organs/weight.
Description
Technical field
The present invention relates to field of traditional Chinese, and in particular to a kind of compound preparation and its system of the tree peony peptide with strengthen immunity
Preparation Method.
Background technology
Today's society, with social competition's fierceness, operating pressure increases, and rhythm of life is getting faster.In daily life, people are past
Toward the reasonably combined of diet is had no time to attend to, immunity degradation is caused.In the period of extraneous influenza etc. is prevailing, such crowd often most holds
It is vulnerable to infection.Modern life keen competition, fast pace, high pressure have aggravated the physical and mental burden of people；It is environmental pollution, bad
Living habit etc., such as eat and drink immoderately, edible phitosite, cause to be deficient in vitamin with mineral matter element etc., from another
Aspect reduces the fitness of people, weakens the immune function of body, these are all the inducements for causing sub-health state to occur.
Therefore, using the various Chinese medicines with immunological enhancement or plant component to improving body's immunity lowly with important
Meaning.
Immune system is the host defence mechanism of human body, is any foreign matter (virus, bacterium that body identified and eliminated external intrusion
Deng)；With identify and processing aging, damage, death, mutant ability.The presence of immune system and the normalization of function
It is the basic guarantee that body's immunity is stablized, the defect or exception of an any of which part can all lead to the incomplete of immune function
Or it is disorderly, to reduce or lose immune function.The deficiency of immune function can lowly generate body health totally unfavorable
It influences, multiple infectious disease or the morbidity and mortality of non-infective disease is made to improve.Immunology Today thinks that immunity is that human body is known
Physiological reaction that is other and excluding " dissident ".Sound immune system mainly has three zones：
1, defense function --- protection body is without damage, and body is helped to eliminate external bacterium, virus and avoid that disease occurs
Disease；
2, stablize cleaning function --- the cell of aging death is constantly removed, the purification update in keeping body；
3, monitoring function --- identification in time and the cell for removing chromosome aberration or gene mutation, prevent the hair of tumour and canceration
It is raw.
The immune function of human body is influenced by human body gene mostly, but acquired environment influences also very the immunity function of human body
Greatly, such as：Diet, sleep, movement, pressure etc..Wherein diet has important influence power, because the ingredient of some foods can assist
Stimulation immune system is helped, immune function is enhanced.
The influence to immune function such as Chinese medicine or plant component can be reflected in the promotion to the links of immune system and function,
Inhibition and adjustment effect, such as to immune organ, mononuclear phagocyte system, T cell, B cell and the antibody of generation, red blood cell
Function, NK cells, LAK cells, neutrophil leucocyte, level of complement and complement activity, interleukins, interferon, colony thorn
Swash the factor, tumor necrosis factor, cell adhesion factor, nitric oxide and neuroendocrine immunomodulation etc..
For the prevention of disease, rehabilitation and improve sub-health state with immunoregulatory Chinese medicine or plant component, improves life
Life quality etc. is of great significance.For this purpose, state food drug inspection office, which will clearly adjust immune function, is included in health care food
Within 22 functions of product.
Currently, common immunopotentiator mainly has following three categories：The first kind is more with lentinan, tremella polysaccharides, yeast
Sugar is the Chinese medicine and fungi polysaccharide para-immunity reinforcing agent of representative；Second class is nucleic acid immunopotentiator, the core after their degradations
Thuja acid, deoxynucleotide can be immediately used by the body and absorb；Third class is trace element-necessary to maintain body normal activities
Selenium participates in the adjustment of body different physiological roles in the form of multiple biological activities in vivo.
Invention content
To solve the above problems, the present invention provides a kind of preparation method of the compound preparation of the strengthen immunity of peptide containing tree peony,
Gained preparation is with obvious effects, being capable of strengthen immunity.
To achieve the above object, the technical solution that the present invention takes is：
A kind of compound preparation of the strengthen immunity of the peptide containing tree peony, is prepared by the raw material of following parts by weight：
3-6 parts of dendrobium candidum solid powder, 4-8 parts of Radix Astragali solid powder, 2-4 parts of sealwort solid powder, 6-10 parts of tree peony peptide.
Preferably, the dendrobium candidum solid powder is dendrobium candidum water extract or dendrobium candidum extractive with organic solvent；Institute
It is Radix Astragali water extract or Radix Astragali extractive with organic solvent to state Radix Astragali solid powder；The sealwort solid powder extracts for sealwort water
Object or sealwort extractive with organic solvent；The tree peony peptide solid powder is extract of the tree peony albumen after complex enzyme hydrolysis.
Preferably, the dendrobium candidum solid powder is as obtained by following steps preparation：
After the dendrobium candidum of moisture ＜ 12% is smashed, with the solid-liquid ratio of 1: 10 (g/ml), first with 60 DEG C of hot-water soak
1h boils 5min by intense fire, is cooked by slow fire 1h, and 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations；Merge concentration thick substances, be dried in vacuo under conditions of 60 DEG C to get.
Preferably, the Radix Astragali solid powder is as obtained by following steps preparation：
After the Radix Astragali of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge the thick substances of concentration, be dried in vacuo under conditions of 60 DEG C to get.
Preferably, the sealwort solid powder is as obtained by following steps preparation：
After the sealwort of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge concentration thick substances be dried in vacuo under conditions of 60 DEG C to get.
Preferably, the tree peony peptide solid powder is as obtained by following steps preparation：
After the tree peony dregs of rice of moisture ＜ 15% are smashed, sieving is impregnated 1h with 1: 20 (g/ml) solid-liquid ratio deionized water, is added
Enter by 0.5-2 parts of neutral proteinase, 1-3 parts of papain, 1-2 parts of flavor protease, is 5.0-9.0, temperature 30- in pH
70 DEG C, concentration of substrate 1.0-10%, enzyme concentration is digested under conditions of being 6000-10000U/g, synchronous stirring hydrolysis 6h,
Stir speed (S.S.) is 200-4000r/min, and 5000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations, merge the thick substances of concentration be spray-dried to get.
The present invention also provides a kind of above-mentioned preparation methods of the compound preparation of the strengthen immunity of the peptide containing tree peony, including walk as follows
Suddenly：
S1, each component is weighed by the formula of preceding claim；
S2, it after mixing the dendrobium candidum powder weighed, Radix Astragali solid powder, sealwort solid powder, tree peony Gly-His-Lys, solves solution or divides
It is dispersed in suitable excipient and solution, suspension, lotion or semisolid is made, be sealed in spherical or ellipse soft capsule material
Capsule to get.
Involved medicinal material and its pharmacology are in the present invention：
Dendrobium candidum, Dendrobium officinale Kimuraet Migo are sweet in flavor, cold nature.Return stomach, kidney channel.Function with
It cures mainly：Reinforcing stomach reg fluid, nourishing Yin and clearing heat.To hinder for pyreticosis Tianjin, mouthfeel polydipsia, deficiency of stomach-Yin, deficiency of food is retched, and abnormal heat is not moved back after being ill,
Fire excess from yin deficiency, hectic fever due to yin labor heat, mesh is secretly unknown, muscles and bones impotence.
Radix Astragali, legume, Astragalus membranaceus Bge.Var.monghoicus, sweet in flavor, slightly warm in nature.Function
With cure mainly tonifying Qi and lifting yang, strengthening exterior and reducing sweat, inducing diuresis for removing edema, shengjin nourishing, move stagnation and palsy, pus draining and toxin expelling, expelling pus and promoting granulation.For the deficiency of vital energy
It is weak, anorexia and loose stool, the sinking of qi of middle-jiao, rush down prolapse of the anus for a long time, uterine bleeding of having blood in stool, exterior deficiency spontaneous perspiration, qi deficiency edema, Heat Diabetes, blood deficiency chlorosis,
Hemiplegia, numbness pain is numb, burst for a long time and does not holds back.
Sealwort, Polygonatum sibiricum Red, liliaceous plant is sweet in flavor, mild-natured.Returns spleen stomach kidney channel.It cures mainly：Tonifying Qi
Yin-nourishing, invigorating the spleen moistening lung, kidney-nourishing.For deficiency of spleen-QI and stomach-QI, fatigue and asthenia, deficiency of stomach-Yin, dry deficiency of food, deficiency syndrome of the lung cough caused by dryness, overstrain cough hemoptysis,
Asthenia of essence and blood, soreness and weakness of waist and knees, poliosis, Heat Diabetes.
Tree peony peptide, the tree peony dregs of rice after tree peony squeezing, obtains after a variety of biological enzyme composite hydrolysis, has enhancing human immunity
The bioactivity of power.
The invention has the advantages that：
It is proved through animal experiment, tree peony peptide compound preparation provided by the present invention has the function of strengthen immunity.Orally administration
The composition of mouse various dose 30 days can enhance the conversion capability of the mouse spleen lymphocyte of ConA inductions, delayed becomes
State is reacted, and cellular immune function can be enhanced；The carbonic clearance ability that mouse monokaryon-macrophage can be improved, can enhance monokaryon-
Macrophage function；The ability for promoting mouse antibodies cellulation, can promote mouse humoral immune；It can promote mouse abdomen
Chamber macrophage swallows the phagocytic activity of fluorescent microsphere.On the body weight increase of mouse, internal organs/weight ratio without influence.
Specific implementation mode
In order to make objects and advantages of the present invention be more clearly understood, the present invention is carried out with reference to embodiments further detailed
Explanation.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not intended to limit the present invention.
Embodiment 1
A kind of compound preparation of the strengthen immunity of the peptide containing tree peony, is prepared by the raw material of following parts by weight：
3-6 parts of dendrobium candidum solid powder, 4-8 parts of Radix Astragali solid powder, 2-4 parts of sealwort solid powder, 6-10 parts of tree peony peptide；Institute
Dendrobium candidum solid powder is stated as obtained by following steps preparation：
After the dendrobium candidum of moisture ＜ 12% is smashed, with the solid-liquid ratio of 1: 10 (g/ml), first with 60 DEG C of hot-water soak
1h boils 5min by intense fire, is cooked by slow fire 1h, and 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations；Merge concentration thick substances, be dried in vacuo under conditions of 60 DEG C to get.
The Radix Astragali solid powder is as obtained by following steps preparation：
After the Radix Astragali of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge the thick substances of concentration, be dried in vacuo under conditions of 60 DEG C to get.
The sealwort solid powder is as obtained by following steps preparation：
After the sealwort of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge concentration thick substances be dried in vacuo under conditions of 60 DEG C to get.
The tree peony peptide solid powder is as obtained by following steps preparation：
After the tree peony dregs of rice of moisture ＜ 15% are smashed, sieving is impregnated 1h with 1: 20 (g/ml) solid-liquid ratio deionized water, is added
Enter by 0.5-2 parts of neutral proteinase, 1-3 parts of papain, 1-2 parts of flavor protease, is 5.0-9.0, temperature 30- in pH
70 DEG C, concentration of substrate 1.0-10%, enzyme concentration is digested under conditions of being 6000-10000U/g, synchronous stirring hydrolysis 6h,
Stir speed (S.S.) is 200-4000r/min, and 5000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations, merge the thick substances of concentration be spray-dried to get.
Embodiment 2
A kind of preparation method containing sea cucumber, the compound preparation of the strengthen immunity of the stem of noble dendrobium, includes the following steps：
S1, each component is weighed according to the component of following parts by weight：
3-6 parts of dendrobium officinale aqueous extract solid powder, 4-8 parts of Radix Astragali water extract solid powder, sealwort water extract solid powder 2-4
Part, 6-10 parts of sea cucumber water extract solid powder.
S2, the dendrobium candidum powder weighed, Radix Astragali solid powder, sealwort solid powder, tree peony Gly-His-Lys are mixed, by these powder
Solution solution is divided
It is dispersed in suitable excipient and solution, suspension, lotion or semisolid is made, be sealed in spherical or ellipse soft capsule
Capsule in material.Obtain the dendrobium candidum compound preparation of strengthen immunity function.
Preferably, also the component that above-mentioned steps S1 is weighed can be prepared into capsule, granule, piece using conventional molding processes
Agent, powder or pill preparation.
Dendrobium candidum in this specific implementation has the effects that strengthen immunity, anti-oxidant, antitumor, hypoglycemic, blood fat；Radix Astragali
It is medicinal have more than 2000 years history so far, have liver protection, diuresis, anti-aging, resisting stress, strengthen immunity, decompression and extensively
General antibacterial action；Jishengshenqiwan Urine proteins can be eliminated, myocardial contractive power is enhanced, adjust blood-sugar content；Sealwort is sweet in flavor mild-natured,
Enter lung spleen kidney channel, there is kidney tonifying, strengthening the essence, tonifying middle-Jiao and Qi, embellish lung, five viscera settling, only cold and other effects；It lives containing immune in tree peony peptide
Property small peptide, human immunoglobulin's total amount and it is made to maintain high level by improving, the variation of antibody activity is stimulated, to carry
The overall immune ability of high people, and itself conditioning functions of human body can be promoted, in addition, the also work(with anti-oxidant, anti-inflammatory, removing toxic substances
Effect.
The functional evaluation of the tree peony peptide compound preparation of the present invention
1.1 test sample：
After the dendrobium candidum powder weighed in embodiment 1, Radix Astragali solid powder, sealwort solid powder, tree peony Gly-His-Lys are mixed,
Suitable pure water is added and prepares a certain concentration, stirring until be completely dissolved to get.
1.2 experimental animal
The cleaning grade BALB/c male mices provided by Beijing HFK Bio-Technology Co., Ltd., SPF grades, production permit
Card SCXK- (capital) 2014-0004.Feed is provided by Shanghai Slac Experimental Animal Co., Ltd., production licence number：
SCXK- (Shanghai) 2014-0001.
Experimental animal room environmental condition：Animal Lab. is barrier system, zoopery room temperature：22-25 DEG C, relative humidity：
55%-70%.
The setting of 1.3 zoopery dosage
According to《Health food is examined and assessment technique specification》2003 editions (hereinafter referred to as specifications) determine experimental program and dosage.It is real
Test animal recommendation inbred mouse, BALB/c etc., 6~8 week old, 18~22g (BALB/c kinds can 16-18g), male, every group
10.
According to《Pharmacopeia (2010 editions)》In, dendrobium candidum is as the 1/3- that health food recommended dose is pharmacopeia recommended dose
1/2.Therefore in the health food dendrobium candidum dosage 2-6g, the dosage 3-15g of Radix Astragali, the dosage 3-7.5g of sealwort, it is male
4-8 parts of the dosage of red peptide.According to the solid content in formula rate and extraction process, obtains human oral 4.8g/d, change
Calculate animal dosage：Mice clinical equivalent is 2.5g/kg (middle dosage).
1.4 experiment process
It chooses various animals and sets 5 dosage groups：Negative control group, model control group, low dosage (1.25g/kg), middle dosage
(2.5g/kg), high dose group (3.75g/kg), these three dosage groups are respectively equivalent to 5 times, 10 times, 30 times of human body recommended amounts.
Minimum effective dose is found out, one of dosage is equivalent to 10 times (mouse) that human body recommends intake dosage, and maximum dose level does not surpass
Cross human body recommended dose 30 times.Corresponding dosage group animal gavage is given by the volume of 0.2ml/20g.BW, control group gives
The physiological saline of volume, daily gavage is primary, continuous gavage 30 days.Negative control group and HY group gavages give isodose physiology
Brine, the 22nd, 24,26,28 and 30 day, each group intramuscular injection HY 40mg/kg.bw in addition to negative control group, negative control group note
Isometric physiological saline is penetrated, carries out each index test within the 31st day.
1.5 experimental methods and result
1.5.1 weight, internal organs/weight ratio measurement
Mouse is put to death after weighing, takes out thymus gland and spleen, is removed most fascia, organ surface blood stains is blotted with filter paper, in electronic analysis
It weighs on balance, calculates weight, thymus gland/weight ratio, spleen/weight ratio.
As a result：Orally administration tested material 30 days, with negative control group ratio, model group mouse weight significantly reduces (P ＜ 0.01), says
Bright immunosuppression model is successfully prepared.
Influence of the compound preparation of the strengthen immunity of 1 peptide containing tree peony of table to hypoimmunity mice weight
Note：Model group is compared with negative control group, * * P ＜ 0.01, n=10
Influence of the compound preparation of the strengthen immunity of 2 peptide containing tree peony of table to hypoimmunity mice internal organs/weight ratio
Note：Model group is compared with negative control group, and * * P ＜ 0.01, dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
As seen from the above table, it is statistically analyzed, gavage gives tested material 30 days, with negative control group ratio, model group mouse weight
It significantly reduces (P ＜ 0.01), illustrates that immunosuppression model is successfully prepared.Each dosage group mouse weight, weightening and model control group
Between comparing difference without notable (p ＞ 0.05), i.e. tested material has no significant effect mouse weight, weightening.
1.5.2 the mouse spleen lymphocyte transformation experiment (mtt assay) of ConA inductions
It is sterile to take spleen, it is placed in and fills in appropriate (3-5ml) sterile Hank ' s liquid plates, and one piece of gauze is placed above in spleen, use
Large syringe inner core gently grinds spleen, and individual cells suspension is made.It filters through 200 mesh screens, is washed 2 times with Hank ' s liquid,
Centrifugation 10min (1000rpm) every time.Then cell is suspended in the complete culture solution of 2ml, is lived with platform phenol orchid dyeing counting thin
Born of the same parents' number (should be 95% or more), adjustment cell concentration are 3 × 106A/ml.
Holes is divided to be added in 24 well culture plates cell suspension, per hole 1ml, a hole adds 75 μ l ConA liquid (to be equivalent to 7.5 μ g/
Ml), 5%CO as a contrast, is set in another hole2, 37 DEG C, CO272h is cultivated in incubator.Culture terminates preceding 4h, is gently sucked per hole
Clear liquid 0.7ml is added 0.7ml and is free of the RPMI1640 culture solutions of calf serum, while 50 holes μ l/ MTT (5mg/ml) are added, after
Continuous culture 4h.After culture, add 1ml acid isopropyl alcohol per hole, ultrasonic vibration (2s) or manually blow and beat mixing, makes purple crystal
It is completely dissolved.Then it being dispensed into 96 well culture plates, each hole dispenses 3 holes as Duplicate Samples, with enzyme-linked immunosorbent assay instrument, with
570nm wavelength measures OD value.Also lysate can directly be moved into 2ml cuvettes, in wavelength 570nm on spectrophotometer
Measure OD values.Proliferation rate=(ODExperimental group/ODBlank control group-1)
The compound preparation of the strengthen immunity of 3 peptide containing tree peony of table is to the ConA hypoimmunity mice spleen lymphocyte proliferations induced
It influences
Note：Model group is compared with negative control group, and * * P ＜ 0.01, dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
As seen from the above table, it is statistically analyzed, gavage gives tested material 30 days, with negative control group ratio, model group mouse weight
It significantly reduces (P ＜ 0.01), illustrates that immunosuppression model is successfully prepared.Middle and high dosage group can promote mice spleen lymph respectively
The proliferation of cell.The effect of middle dose group is extremely notable.
1.5.3 delayed allergy (DTH)
Mouse skin of abdomen is lost hair or feathers with barium sulphide, range about 3cm × 3cm, uniformly smears sensitization with 50 μ l of DNFB solution.After 5 days, use
10 μ l of DNFB solution are uniformly applied to mouse right ear (two sides) and are attacked.Cervical dislocation puts to death mouse for 24 hours after attack, cuts a left side
Auris dextra shell.The auricle that diameter 8mm is removed with card punch, weighs.The degree of DTH is indicated with the difference of left and right ear weight.Given the test agent
The weight difference of group is significantly higher than the weight difference with control group, can determine that this experimental result positive.
Influence of the compound preparation of the strengthen immunity of 4 peptide containing tree peony of table to hypoimmunity mice ear swelling degree
Note：Model group is compared with negative control group, and * * P ＜ 0.01, dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
As seen from the above table, it is statistically analyzed, gavage gives tested material 30 days, with negative control group ratio, model group mouse weight
It significantly reduces (P ＜ 0.01), illustrates that immunosuppression model is successfully prepared.The ear swelling degree of middle and high dosage group is significantly higher than low dose
Amount group and negative control group.
1.5.4 mouse carbonic clearance is tested
It after culture, weighs, injects diluted india ink from mouse tail vein, calculated by per 10g weight 0.1ml.It waits for
Prepared Chinese ink injects, timing immediately.Inject prepared Chinese ink after 2,10min, take 20 μ l of blood from angular vein clump respectively, exist side by side be added into
In 2ml 0.1%Na2CO3 solution.With spectrophotometer or full-automatic microplate reader at 600nm wavelength densitometric value (OD),
With Na2CO3Solution makees blank control.
Mouse is put to death, liver and spleen are taken, organ surface blood stains is blotted with filter paper, weighs.
The ability of mouse carbonic clearance is indicated with phagocytic index.Phagocytic index a is calculated as follows：
Influence of the compound preparation of the strengthen immunity of 5 peptide containing tree peony of table to hypoimmunity mice carbonic clearance phagocytic function
Note：Model group is compared with negative control group, * P ＜ 0.01, and dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
By table 5 as it can be seen that HY group mouse carbonic clearance phagocytic functions are below negative control group, difference has statistical significance (P ＜
0.01).For each dosage group of dendrobium candidum compound preparation compared with HY groups, phagocytic index has raised trend, and middle dose group
Effect is preferable.
1.5.5 antibody-producting cell detection (improved method)
It is prepared by SRBC
Sheep blood is put into the sterilizing conical flask of bead, shakes in one direction by sheep taking blood from jugular vein, to take off fiber,
Physiological saline 2500rpm, 15min are washed 3 times, are prepared hematocrit SRBC, be put into 4 DEG C of refrigerators and save backup.
The preparation of complete medium
Newborn calf serum through sheep red blood cell (SRBC) (v/v, 5: 1) absorbing, 4 DEG C, after 30-60min, incomplete culture medium RPMI is added
In 1640, it is prepared into the complete medium containing 20% calf serum.
It is prepared by complement
Femoral artery takes blood, stands 30-60min, and 2500rpm, 15min detach serum, after 5-10 guinea pig serum is mixed, with pressure
Product SRBC mix with 5: 1 (v/v) ratios, and 4 DEG C of refrigerators place 30-60min, or concussion, 2500rpm, 15min separation serum,
Packing, -80 DEG C of refrigerators preserve.Used time is with complete medium 1: 10 (v/v) dilution proportion.
Immune animal
The cell suspension (about 1 × 108 SRBC) of 2% (v/v), every mouse intraperitoneal injection are made with physiological saline by hematocrit SRBC
0.2ml。
It is prepared by splenocyte suspension
Mouse cervical dislocation after SRBC is immunized 4-5 days is put to death, sterile to take spleen, and one piece of gauze is placed above in spleen, is used
Large syringe inner core gently grinds spleen, individual cells suspension is made, the filtering of 200 mesh screens, 1000rpm, 10min are gone
Clearly, Hank ' liquid is washed 2 times, and 5 × 106~1 × 10 are prepared into complete medium RPMI 16407The splenocyte suspension of a/ml.
The measurement of plaque
It is prepared by bottom culture medium：0.5g agaroses are added in 100ml sterile salines, dissolve by heating, wait for temperature in 50 DEG C of left sides
When right, added in six well culture plates with the amount in the holes 1ml/, it is spare after agar solidification.
It is prepared by top layer culture medium：0.5g agaroses are added in 100ml Hank ' s liquid (PH 7.2-7.4), dissolve by heating, with
The amount of 0.5ml/ test tubes adds in the test tube of 46-50 DEG C of constant temperature.
Bed board：50 μ l, 20%SRBC (normal saline, v/v), 200 μ l splenocyte suspensions are successively added and contain 0.5ml top layers
Test tube in, top layer mixed liquor is poured into six orifice plates and paved to rapid mixing, and each sample does two parallel holes.
Plaque assay：It has prepared culture plate and has been put into 37 DEG C, 5%CO21h is incubated in incubator, then per hole be added 500 μ l with
The diluted complement of complete medium (v/v, 1: 10), continuing to be incubated 2h, automatic image analyzer reads hemolysis plaque graphical analysis
Software counts hemolysis plaque number.It is the hemolysis plaque numerical value of sample to take the average value of parallel hole plaque number, with plaque number/106Spleen
Cell indicates.
The compound preparation of the strengthen immunity of 6 peptide containing tree peony of table is to hypoimmunity mice hemolysin, plaque forming cells's situation
It influences
Note：Model group is compared with negative control group, and * * P ＜ 0.01, dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
Table 6 the result shows that, with blank negative control group ratio, model group hemolysin and plaque forming cells's situation significantly reduce (P
＜ 0.01), illustrate that immunosuppression model is successfully prepared.With model group ratio, in, low dosage can remarkably promote mouse hemolysin and
The generation (P ＜ 0.01) of plaque forming cells.
1.5.6 Turnover of Mouse Peritoneal Macrophages phagocytosis fluorescent microsphere experiment
Experiment injects 0.2ml in first 4 days to every mouse peritoneal, 2% sheep erythrocyte activates mouse macrophage, experimental day
Hank ' s liquid 3ml/ that mouse, intraperitoneal injection plus calf serum are put to death with cervical dislocation, gently rubs abdomen 20 times, to fill
Divide and wash out peritoneal macrophage, stomach wall is then cut off into an osculum, draws abdominal cavity washing lotion 2ml and filtered to examination with 75 μm of filters
In pipe, adjustment number of macrophages are 4~6 × 105/ml.1ml abdominal cavity washing lotions are drawn in 6 well culture plates with liquid-transfering gun, are added
The fluorescent microsphere (1 × 10 crossed through preconditioned7/ plate), 37 DEG C of carbon dioxide cell incubators (or room temperature) are protected from light incubation 90~120
Minute, supernatant (containing non-attached cell and extra fluorescent microsphere) is abandoned after incubation, is gently washed using 1ml, PBS buffer solution every time
It washs 2 times, adds 4 DEG C of PBS buffer solution 0.3ml after removing supernatant, attached cell is scraped with cell scraper, gently through 75 μm after piping and druming uniformly
Upper machine analysis after filter filtering.
It is analyzed using flow cytomery, data processing and result judgement
The phagocytic activity of mouse macrophage is indicated with phagocytic percentage or phagocytic index.Phagocytic percentage need to carry out data and turn
It changes,P is phagocytic percentage in formula, decimally indicates, then carries out variance analysis again.Test sample group gulps down
Percentage, phagocytic index are bitten compared with control group phagocytic percentage, phagocytic index, difference is significant, can determine that this reality
Test the result positive.
Influence of the compound preparation of the strengthen immunity of 7 peptide containing tree peony of table to hypoimmunity mice Peritoneal Macrophage Phagocytosis
Note：Model group is compared with negative control group, * P ＜ 0.01, and dosage group is compared with HY groups, * P ＜ 0.05, * * P ＜ 0.01；n
=10
Table 7 the result shows that：With blank control group ratio, model group Turnover of Mouse Peritoneal Macrophages phagocytic percentage significantly reduces (P ＜
0.01), phagocytic index is substantially reduced (P ＜ 0.05), illustrates that immunosuppression model is successfully prepared.With model group ratio, middle dosage point
Not can pole significantly improve phagocytic percentage (P ＜ 0.01) of the immunosuppressed mice peritoneal macrophage to fluorescent microsphere, low dose
Amount group is remarkably improved peritoneal macrophage phagocytic index (P ＜ 0.01).
Experimental result
1, mouse weight, internal organs/weight ratio
The mouse weight of negative control group, the ratio of internal organs/weight are all remarkably higher than this two indexs of model group mouse, table
Bright modeling success.Each dosage group is compared with model group more without significant difference, and therefore, which does not interfere with the body of mouse
Weight and weight/internal organs ratio.
2, cell immunization experiments project
In mouse lymphocyte proliferation experiment, in the compound preparation of the tree peony peptide of orally administration mouse various dose, high agent
Amount group mice spleen lymph conversion capability is apparently higher than negative control group, and difference has conspicuousness (P ＜ 0.05), and middle dose group has
Extremely significant difference, effect is preferable, and shows that the sample has lymphopoiesis, the conversion capability for promoting mouse.
In ear swelling experiment, the compound preparation of the tree peony peptide of orally administration mouse various dose, middle and high dosage group mouse
The weight difference of left and right auricle is slightly far above model group, is also apparently higher than blank control group.Show that the sample can promote mouse
Delayed allergy.
3, humoral immunity experimental project
In the tree peony peptide compound preparation of orally administration mouse various dose, in, the antibody cell of the mouse of low dosage generate it is thin
Born of the same parents' number is higher than model group, blank control group.With conspicuousness.Show that the sample has and promotes mouse antibodies cellulation proliferation
Effect.
4, mononuclear-macrophage phagocytic function
In the carbonic clearance experiment of mouse, the tree peony peptide compound preparation of orally administration mouse various dose is high, middle dose group
The phagocytic index of mouse is higher than control group, has apparent difference with blank control group, shows that the sample has the list for promoting mouse
The carbonic clearance ability of core-macrophage.
It presses《Specification》It is required that in cellular immune function, humoral immune function, one macrophage function of monokaryon, NK cell activity four
Any two aspect results of a aspect are positive, can determine that the given the test agent has strengthen immunity function.Therefore, the above results
Show that the present invention is that have the function of strengthen immunity, especially not only dosage is few for middle dose group, but also with relatively good
Strengthen immunity function.
In conclusion said preparation is suitable for immunocompromised person, has effects that strengthen immunity.Mice spleen can not only be promoted to drench
Proliferation transformation, the monocytes/macrophages carbonic clearance ability of bar cell, while the ability of antibody-producting cell is also improved, promote
Into the delayed allergy of immunodeficiency models mouse caused by anti-hydrocortisone.In addition, to weight, internal organs/weight
Ratio has not significant impact.
The above is only a preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, without departing from the principle of the present invention, can also make several improvements and retouch, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (7)
1. a kind of compound preparation of the strengthen immunity of peptide containing tree peony, which is characterized in that by the raw material preparation of following parts by weight
At：
3-6 parts of dendrobium candidum solid powder, 4-8 parts of Radix Astragali solid powder, 2-4 parts of sealwort solid powder, 6-10 parts of tree peony peptide.
2. a kind of compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, which is characterized in that the iron sheet
Stem of noble dendrobium solid powder is dendrobium candidum water extract or dendrobium candidum extractive with organic solvent；The Radix Astragali solid powder is Radix Astragali
Water extract or Radix Astragali extractive with organic solvent；The sealwort solid powder is that sealwort water extract or sealwort organic solvent extract
Object；The tree peony peptide solid powder is extract of the tree peony albumen after complex enzyme hydrolysis.
3. a kind of compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, which is characterized in that the iron sheet
Stem of noble dendrobium solid powder is as obtained by following steps preparation：
After the dendrobium candidum of moisture ＜ 12% is smashed, with the solid-liquid ratio of 1: 10 (g/ml), first with 60 DEG C of hot-water soak
1h boils 5min by intense fire, is cooked by slow fire 1h, and 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations；Merge concentration thick substances, be dried in vacuo under conditions of 60 DEG C to get.
4. a kind of compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, which is characterized in that the Radix Astragali
Solid powder is as obtained by following steps preparation：
After the Radix Astragali of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge the thick substances of concentration, be dried in vacuo under conditions of 60 DEG C to get.
5. a kind of compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, which is characterized in that the sealwort
Solid powder is as obtained by following steps preparation：
After the sealwort of moisture ＜ 12% is smashed, with 1: 10 (g/ml) solid-liquid ratio, first with 60 DEG C of hot-water soak 1h, intense fire
5min is boiled, 1h is cooked by slow fire, 3000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats above-mentioned behaviour
Make, merge concentration thick substances be dried in vacuo under conditions of 60 DEG C to get.
6. a kind of compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, which is characterized in that the tree peony
Peptide solid powder is as obtained by following steps preparation：
After the tree peony dregs of rice of moisture ＜ 15% are smashed, sieving is impregnated 1h with 1: 20 (g/ml) solid-liquid ratio deionized water, is added
Enter by 0.5-2 parts of neutral proteinase, 1-3 parts of papain, 1-2 parts of flavor protease, is 5.0-9.0, temperature 30- in pH
70 DEG C, concentration of substrate 1.0-10%, enzyme concentration is digested under conditions of being 6000-10000U/g, synchronous stirring hydrolysis 6h,
Stir speed (S.S.) is 200-4000r/min, and 5000rpm centrifuges 10min later, and suction filtration takes filtrate to be concentrated under reduced pressure, and filter residue repeats
Aforesaid operations, merge the thick substances of concentration be spray-dried to get.
7. a kind of preparation method of the compound preparation of the strengthen immunity of the peptide containing tree peony as described in claim 1, feature exist
In including the following steps：
S1, each component is weighed by claim 1-6 any one of them formulas；
S2, it after mixing the dendrobium candidum powder weighed, Radix Astragali solid powder, sealwort solid powder, tree peony Gly-His-Lys, solves solution or divides
It is dispersed in suitable excipient and solution, suspension, lotion or semisolid is made, be sealed in spherical or ellipse soft capsule material
Capsule to get.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102160615A (en) * | 2011-02-24 | 2011-08-24 | 洛阳红娇天香牡丹生物有限公司 | Health-care food component taking peony extract as base material and application thereof |
| CN102600368A (en) * | 2012-03-21 | 2012-07-25 | 四川万安石斛产业开发有限公司 | Pharmaceutical composition, as well as preparation method and application thereof |
| CN104041831A (en) * | 2014-06-26 | 2014-09-17 | 河南科技大学 | Soft peony peptide capsule and preparation method thereof |
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2018
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| CN102160615A (en) * | 2011-02-24 | 2011-08-24 | 洛阳红娇天香牡丹生物有限公司 | Health-care food component taking peony extract as base material and application thereof |
| CN102600368A (en) * | 2012-03-21 | 2012-07-25 | 四川万安石斛产业开发有限公司 | Pharmaceutical composition, as well as preparation method and application thereof |
| CN104041831A (en) * | 2014-06-26 | 2014-09-17 | 河南科技大学 | Soft peony peptide capsule and preparation method thereof |
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Application publication date: 20180817 |