CN110478470A - A kind of anti-gout composition of the small-molecular peptides containing sunflower disk and preparation method thereof - Google Patents
A kind of anti-gout composition of the small-molecular peptides containing sunflower disk and preparation method thereof Download PDFInfo
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- CN110478470A CN110478470A CN201910803114.2A CN201910803114A CN110478470A CN 110478470 A CN110478470 A CN 110478470A CN 201910803114 A CN201910803114 A CN 201910803114A CN 110478470 A CN110478470 A CN 110478470A
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Classifications
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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
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Abstract
The invention discloses anti-gout compositions of a kind of small-molecular peptides containing sunflower disk and preparation method thereof, raw material and its parts by weight for preparing the drug are as follows: 10~30 parts of sunflower disk, 20~60 parts of semen coicis, 20~60 parts of lophatherum gracile, 20~60 parts of rhizoma imperatae, 20~60 parts of eucommia Bark male flower, 20~60 parts of hawthorn, the raw material that the anti-gout composition of present invention small-molecular peptides containing sunflower disk is selected is the Chinese medicine of integration of drinking and medicinal herbs, sunflower disk small-molecular peptides are shared with these Chinese medical extracts, synergistic effect is good, and it has no toxic side effect, clearing damp and promoting diuresis, promoting blood circulationization addiction, clearing heat-fire, consolidate spleen kidney, urarthritis can be treated by anti-trioxypurine, and have anti-inflammatory, analgesic effect, solves the big puzzlement of current western medicine gout bring side effect, it can take for a long time, reduce uric acid, from And treat gout.
Description
Technical field
The present invention relates to a kind of anti-gout compositions and preparation method thereof, and in particular to a kind of small-molecular peptides containing sunflower disk
Anti-gout composition and preparation method thereof belongs to Chinese medicine pharmaceutical technology field.
Background technique
Gout (Gout) is a kind of due to purine metabolic disturbance, and the final product uric acid content of purine metabolism is caused to increase extremely
Caused metabolic disease.Gout is characterized in apparent hyperuricemia, and lithate monohydrate crystal is in joint and kidney
Middle deposition, leads to urarthritis and uric acid kidney stones.Hyperuricemia (Hyperui~cemia) can be considered as the various hearts
The clinically important risk factor of vascular diseases.And hyperuricemia and cancer, obesity, insulin resistance, hypertension and blood
Rouge abnormal (metabolic syndrome) is also related.Strict control uric acid level be the key that prevention gout and other related diseases because
Element.
Sunflower disk has a variety of curative effects of anti-trioxypurine, antitumor, anti-oxidant, antianginal, antibacterial, analgesia, blood pressure lowering etc.,
It is significant in efficacy in addition, the extracted obtained small-molecular peptides of sunflower disk are fast with more effect, the features such as dosage is small；Semen coicis, nature and flavor
It is sweet light slightly cold, there are inducing diuresis for removing edema, invigorating spleen to remove dampness, relaxing muscles and tendons eliminating impediment, clearing away heat and eliminating pus and other effects；Lophatherum gracile has refrigerant, antipyretic, diuresis
And the benefits of hastening parturition；Rhizoma imperatae, gas is micro-, slightly sweet, cool blood, hemostasis, heat-clearing, diuresis；Eucommia Bark male flower, for " strong liver, is led at kidney tonifying
Just, sleeping, lower hyperlipidemia, hypertension, hyperglycemia " effect it is especially pronounced；Hawthorn, taste is slightly sour puckery, has reducing blood lipid, blood pressure, heart tonifying, arrhythmia etc.
Effect.
Anti-gout drugs are mostly based on Western medicine at present, such as colchicin, Indomethacin, prednisolone, allopurinol, non-
Cloth sotan, probenecid, Benzbromarone etc., generally existing toxic side effect and adverse reaction, therefore good effect, poison are found from plant
The small natural active matter of property, is one of current researcher urgent problem to be solved.
Summary of the invention
In view of this, the present invention provides anti-gout composition of a kind of small-molecular peptides containing sunflower disk and preparation method thereof,
The raw material that the composition is selected is dual-purpose of drug and food, and takes the Chinese medicine of toxic side effect, and a few taste collaborations share clearing damp and promoting diuresis, promoting blood circulationization
Addiction, clearing heat-fire consolidate spleen kidney, have significant anti-trioxypurine effect, thus to the curative effect to treatment gout.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of anti-gout composition of the small-molecular peptides containing sunflower disk, raw material and its parts by weight are as follows: sunflower disk 10~
30 parts, 20~60 parts of semen coicis, 20~60 parts of lophatherum gracile, 20~60 parts of rhizoma imperatae, 20~60 parts of eucommia Bark male flower, hawthorn 20~60
Part；
Preferably, parts by weight are as follows: 20 parts of sunflower disk, 40 parts of semen coicis, 40 parts of lophatherum gracile, 40 parts of rhizoma imperatae, Cortex Eucommiae
40 parts of male flower, 40 parts of hawthorn.
The anti-gout composition material combination of present invention small-molecular peptides containing sunflower disk is according to as follows:
Sunflower disk pharmacological property is sweet, cold, return liver warp, can be calming the liver to stop bleeding, clearing away heat to and alleviating pain, can be used for treating hypertension, dizzy head
Bitterly, the diseases such as tinnitus, uterine hemorrhage and sore rash.Modern pharmacological studies have shown that sunflower disk have anti-trioxypurine, it is antitumor, anti-oxidant,
A variety of pharmacological activity such as antianginal, antibacterial, analgesia, blood pressure lowering.
Semen coicis slightly sweet flavor is slightly cold, has inducing diuresis for removing edema, invigorating spleen to remove dampness, relaxing muscles and tendons eliminating impediment, clearing away heat and eliminating pus and other effects, is common
Damp-clearing drug.Adlay is a kind of beauty food again, and often feeding can keep human skin gloss fine and smooth, eliminate acne, freckle,
Senile plaque, cyasma, butterfly macules have good efficacy to furfur, acne, chap, pachylosis etc..
Henon bamboo blade root is medicinal, there is refrigerant, antipyretic, diuresis and the benefits of hasten parturition；The major functions: in the heart disease heat, cough with dyspnea, haematemesis,
Heat toxin wind, only quench one's thirst, press borax poison, dissolving phlegm, control it is fanatical unhappiness, apoplectic aphasia in silence, pain intermittent headache, stop palpitate with fear, pestilence be confused it is bored, kill
Small worm delays spleen except hot.
Rhizoma imperatae gas is micro-, slightly sweet.The major functions: cooling blood and hemostasis, clearing heat and detoxicating.For spitting blood, hematuria, heat gonorrhea, oedema, Huang
Subcutaneous ulcer, difficult urination, pyreticosis and polydipsia, stomach hot vomiting, cough.
Eucommia Bark male flower, Cortex Eucommiae are the trees of the double strains of male and female, and male flower is exactly the flower that Cortex Eucommiae Xiong Shu is opened, scientific research discovery: " Cortex Eucommiae
" spy contains more than 60 kinds of effective plant components, such as lignin, iridoids, Phenylpropanoid Glycosides class active material (such as geniposide to male flower
Acid, chlorogenic acid, Aucubin), rosin spirit disaccharide glucoside, flavones, alkaloid, amino acid, polysaccharide and mineral element Zn, Mn,
Cu, Fe, Ca, P, B, Mg, K etc., eucommia Bark male flower summarize the essence of all Cortex Eucommiaes, it may be said that eucommia Bark male flower has plant Cortex Eucommiae
All health-care efficacies, and it is especially pronounced for the effect of " strong liver, kidney tonifying, defaecation, sleeping, lower hyperlipidemia, hypertension, hyperglycemia ".
The raw material that the anti-gout composition of present invention small-molecular peptides containing sunflower disk is selected is the Chinese medicine of integration of drinking and medicinal herbs, sunflower
Disk small-molecular peptides are shared with these Chinese medical extracts, and synergistic effect is good, and has no toxic side effect, clearing damp and promoting diuresis, promoting blood circulationization addiction, clearly
Hot purging intense heat consolidates spleen kidney, urarthritis can be treated by anti-trioxypurine, and have anti-inflammatory, analgesic effect, solve mesh
The big puzzlement of preceding western medicine gout bring side effect, can take for a long time, uric acid be reduced, to treat gout.
The present invention also provides the preparation methods of the anti-gout composition of the above-mentioned small-molecular peptides containing sunflower disk, and steps are as follows:
(1) each raw material is weighed by above-mentioned parts by weight；
(2) sunflower disk is taken to be soaked in 10~20 times of sunflower disk quality of water, flash extracter extracts 5~15 times, extracts
Liquid is centrifuged 10~30min, takes supernatant, complex enzyme zymohydrolysis, is obtained by filtration the extracting solution that molecular weight is less than 10kd, and temperature -40~
50 DEG C, 0.05~0.08kpa of pressure is freeze-dried to obtain sunflower disk small-molecular peptides freeze-dried powder；
(3) after taking semen coicis to add 10~20 times of semen coicis quality of 0.5~1h of water immersion, 1~3h of extractor refluxing extraction
Obtain semen coicis extracting solution；
(4) after taking lophatherum gracile to add 10~20 times of henon bamboo leaf quality of 0.5~1h of water immersion, 1~3h of extractor refluxing extraction
Obtain henon bamboo leaf extract；
(5) after taking rhizoma imperatae to add 10~20 times of rhizoma imperatae quality of 0.5~1h of water immersion, 1~3h of extractor refluxing extraction
Obtain rhizoma imperatae extracting solution；
(6) after taking eucommia Bark male flower to add 10~20 times of eucommia Bark male flower quality of 0.5~1h of water immersion, extractor refluxing extraction 1
~3h obtains eucommia Bark male flower extracting solution；
(7) after taking hawthorn to add 10~20 times of hawthorn quality of water immersion, 1~3h of extractor refluxing extraction obtains hawthorn extraction
Liquid；
(8) semen coicis extracting solution, henon bamboo leaf extract, rhizoma imperatae extracting solution, eucommia Bark male flower extracting solution and hawthorn are extracted
Liquid mixing, obtains dried extract powder after spray drying；
(9) by sunflower disk small-molecular peptides freeze-dried powder and dried extract powder after mixing up to containing small point of sunflower disk
The anti-gout composition of sub- peptide.
Further, with flash extracter extraction 5~15 times operations in step (2), the time extracted every time is 2~10s.
Further, complex enzyme is the mixed enzyme of pepsin and trypsase, and the temperature of enzymatic hydrolysis is 35~37 DEG C, enzymatic hydrolysis
Time is 4~8h.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
The preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk, steps are as follows:
Step 1: 30 parts of sunflower disk, 15 times of amount water, flash extracter extracts 5 times, extracts 10 seconds every time.Extracting solution from
The heart 10 minutes, supernatant is taken, complex enzyme zymohydrolysis crosses ultrafiltration apparatus, and obtaining molecular weight is respectively to be greater than 10kd, 10kd, is less than
The extracting solution of 10kd.Molecular weight is less than to the extracting solution of 10kd, is dried through medicinal vacuum freeze drier, obtains sunflower disk
Small-molecular peptides freeze-dried powder；
Step 2:, being added 15 times of amount water by 60 parts of semen coicis, impregnates 0.5h, and refluxing extraction 3h in extractor obtains semen coicis and mentions
Take liquid；
Step 3:, being added 15 times of amount water by 60 parts of lophatherum gracile, impregnates 0.5h, and refluxing extraction 3h in extractor obtains lophatherum gracile and mentions
Take liquid；
Step 4:, being added 15 times of amount water by 60 parts of rhizoma imperatae, impregnates 0.5h, and refluxing extraction 3h in extractor obtains rhizoma imperatae and mentions
Take liquid；
Step 5:, being added 15 times of amount water by 60 parts of eucommia Bark male flower, impregnates 0.5h, refluxing extraction 3h in extractor, obtains Cortex Eucommiae hero
Flower extracting solution；
Step 6:, being added 15 times of amount water by 60 parts of hawthorn, impregnates 0.5h, refluxing extraction 3h in extractor, obtains hawthorn extraction
Liquid；
Step 7: step 2 to six extracting solutions is merged, and spray-dried instrument is spray-dried, and dried extract powder is obtained
End；
Step 8: step 1 is uniformly mixed with powder obtained by step 7, obtains the antigout group of the small-molecular peptides containing sunflower disk
Close object.
Embodiment 2
The preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk, steps are as follows:
Step 1: 20 parts of sunflower disk, 10 times of amount water, flash extracter extracts 10 times, extracts 5 seconds every time.Extracting solution from
The heart 30 minutes, supernatant is taken, complex enzyme zymohydrolysis crosses ultrafiltration apparatus, and obtaining molecular weight is respectively to be greater than 10kd, 10kd, is less than
The extracting solution of 10kd.Molecular weight is less than to the extracting solution of 10kd, is dried through medicinal vacuum freeze drier, obtains sunflower disk
Small-molecular peptides freeze-dried powder；
Step 2:, being added 10 times of amount water by 40 parts of semen coicis, impregnates 1h, refluxing extraction 2h in extractor, obtains semen coicis extraction
Liquid；
Step 3:, being added 10 times of amount water by 40 parts of lophatherum gracile, impregnates 1h, refluxing extraction 2h in extractor, obtains lophatherum gracile extraction
Liquid；
Step 4:, being added 10 times of amount water by 40 parts of rhizoma imperatae, impregnates 1h, refluxing extraction 2h in extractor, obtains rhizoma imperatae extraction
Liquid；
Step 5:, being added 10 times of amount water by 40 parts of eucommia Bark male flower, impregnates 1h, and refluxing extraction 2h in extractor obtains eucommia Bark male flower
Extracting solution；
Step 6:, being added 10 times of amount water by 40 parts of hawthorn, impregnates 1h, and refluxing extraction 2h in extractor obtains hawthorn extract；
Step 7: step 2 to six extracting solutions is merged, and spray-dried instrument is spray-dried, and dried extract powder is obtained
End；
Step 8: step 1 is uniformly mixed with powder obtained by step 7, obtains the antigout group of the small-molecular peptides containing sunflower disk
Close object.
Embodiment 3
The preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk, steps are as follows:
Step 1: 10 parts of sunflower disk, 20 times of amount water, flash extracter extracts 15 times, extracts 8 seconds every time.Extracting solution from
The heart 30 minutes, supernatant is taken, complex enzyme zymohydrolysis crosses ultrafiltration apparatus, and obtaining molecular weight is respectively to be greater than 10kd, 10kd, is less than
The extracting solution of 10kd.Molecular weight is less than to the extracting solution of 10kd, is dried through medicinal vacuum freeze drier, obtains sunflower disk
Small-molecular peptides freeze-dried powder；
Step 2:, being added 20 times of amount water by 20 parts of semen coicis, impregnates 1h, refluxing extraction 1h in extractor, obtains semen coicis extraction
Liquid；
Step 3:, being added 20 times of amount water by 20 parts of lophatherum gracile, impregnates 1h, refluxing extraction 1h in extractor, it obtains lophatherum gracile and mentions
Take liquid；
Step 4:, being added 20 times of amount water by 20 parts of rhizoma imperatae, impregnates 1h, refluxing extraction 1h in extractor, it obtains rhizoma imperatae and mentions
Take liquid；
Step 5:, being added 20 times of amount water by 20 parts of eucommia Bark male flower, impregnates 1h, refluxing extraction 1h in extractor, obtain Cortex Eucommiae hero
Flower extracting solution；
Step 6:, being added 20 times of amount water by 20 parts of hawthorn, impregnates 1h, refluxing extraction 1h in extractor, obtain hawthorn extract；
Step 7: step 2 to six extracting solutions is merged, and spray-dried instrument is spray-dried, and dried extract powder is obtained
End；
Step 8: step 1 is uniformly mixed with powder obtained by step 7, obtains the antigout group of the small-molecular peptides containing sunflower disk
Close object.
Embodiment 4
One, in the embodiment of the present invention 1 anti-gout composition of the small-molecular peptides containing sunflower disk to Human umbilical vein endothelial cells
The influence of increment effect
1 materials and methods
1.1 laboratory apparatus and material
C02Incubator, low temperature low speed centrifuge (Guan Guo Hemus company)；Superclean bench (the limited public affairs of Suzhou cleaning equipment
Department)；Electric heating constant-temperature water-bath tank (the upper macro experimental facilities Co., Ltd of Nereid)；Inverted microscope (Japanese Olympus company)；It is micro
Supercentrifuge (LahNet company)；Full-automatic microplate reader (Bio~Red, USA)；Cell incubator (NAPCO, USA)；Streaming
Cell instrument (BD, the U.S.).
DM EM cell culture fluid (GBICO company)；0.25% trypsase (Nanjing Sheng Xing Technology Co., Ltd.)；Calf
Serum FBS (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.)；Sough DMSO (analyzing pure, Shanghai Ling Feng chemical industry) in dimethyl Asia；
MTT (AERESCO, the U.S.).
HUVEC (Human umbilical vein endothelial cells) is provided by preclinical medicine institute, Nanjing University of Traditional Chinese Medicine.
The configuration of 1.2 solution
1.2.1 prepared by lithate
5g uric acid adds 100mL distilled water to boil, and 5%NaOH solution tune pH7.4 is added to stir, and Monosodium urate knot is made in cooling crystallization
Crystallization.
1.2.2DMEM prepared by culture solution uric acid salting liquid
The MSU 10mg high-pressure fire bacterium that will be made, adds the DMEM culture solution 10mL without serum, and grinding is made into 1mg/mL's
DMEM solution.When experiment, this solution adds DMEM culture solution to be made into the MSU solution of various concentration DMEM again.
1.3 experimental method
1.3.1HUVEC in vitro culture
HUVEC plants contain 10% calf through 0.25% trypsin digestion through detection of mycoplasma, no mycoplasma contamination, cell
The DMEM culture solution of serum neutralizes, and is centrifuged (1000r/min x 6min), removes supernatant, the DMEM containing 10% calf serum is added to train
Nutrient solution moves into Tissue Culture Flask, puts 37 DEG C, 5%C02Secondary culture in incubator.
1.3.2HUVEC viability examination
HUVEC is cultivated in culture bottle, to be grown to when 70%~80% fusion, with 0.25% trypsin digestion, is used
10% calf serum DMEM culture solution is tuned into 4x104/ mL cell suspension is implanted into 96 orifice plates (every 200 μ L of hole), and culture for 24 hours, carries out
It tests below, every group of each 8 hole, handles HUVEC with the MSU of final concentration of 0,25,50,75,100 (μ g/mL) respectively, liquid feeding is subsequent
It is continuous to put 37 DEG C, 5%C02It is cultivated in incubator and collects supernatant for 24 hours, remaining HUVEC is for measuring cell activity, every Kong Zaijia
100 μ L of 1mg/mL MTT liquid, continues to put 37 DEG C, 5%C02After cultivating 4h in incubator, MTT liquid is abandoned, dimethyl Asia is added and soughs 200
μ L dissolution, concussion read absorbance value, wavelength 490nm in microplate reader.
2. experimental result
Experimental result is shown in Table 1, and with Normal group cell survival rate (100%) for reference, the present invention contains small point of sunflower disk
When the anti-gout composition of sub- peptide is acted on according to concentration (50 μ g/mL and 100 μ g/mL, 200 μ g/mL), apparent dosage is shown
Dependence, with the increase of dosage, cell survival rate is gradually increased.It is computed, cell survival rate is in 50 μ of concentration after effect for 24 hours
Respectively reach 41.21% ± 0.05%, 47.84% ± 0.02%, 54.34% when g/mL and 100 μ g/mL, 200 μ g/mL ±
0.01%.The result shows that the anti-gout composition of present invention small-molecular peptides containing sunflower disk has certain protection to HUVEC cell
Effect.
Experimental result of the anti-gout composition of 1 small-molecular peptides containing sunflower disk of table to HUVEC cell proliferation rate
| Drug concentration μ g/mL | Appreciation rate |
| 200 concentration | 54.34% |
| 100 concentration | 47.84% |
| 50 concentration | 41.21% |
Two, the anti-gout composition of the small-molecular peptides containing sunflower disk (derives from kidney PC12 cell in the embodiment of the present invention 1
Gland pheochromocytoma) protective effect
1. cell culture processes
The originally culture of 1.1 PC12 cells
With prepared complete culture solution in CO2Routine culture PC12 cell strain is carried out in incubator.CO2In incubator
Temperature, relative humidity, CO2Relative concentration be respectively 37 DEG C, 90%, 5%.Primary above-mentioned culture solution is replaced within every 2 days, and is used
The state of inverted microscope observation group of cells.
The passage of 1.2 PC12 cells
When PC12 cell covers with the state at culture bottle bottom 3/4, illustrate that cell growth is normal and epidemic situation comparison is good.In
In Biohazard Safety Equipment, original culture solution is poured out, the sterile PBS of about 2mL is drawn with Dispette and is shaken back and forth and washed
Cell, repeated washing is twice.0.25% 1~2mL of trypsase is added and is digested (trypsase can cover bottom of bottle), it can be light
Pat and beat culture bottle side, and under inverted microscope the moment check cellular change, when space between cells increases, cell rounding and hair
When raw diffusion, the complete culture solution of same volume is rapidly added to terminate and this time digest, and will be in bottle wall using Dispette
Cell blow and beat completely after, all move into centrifuge tubes in.The revolving speed for adjusting centrifuge is 1000r/min, will be in centrifuge tube
Cell suspension be sufficiently centrifuged after five minutes, suck supernatant.And draw 2mL complete culture solution and be added in centrifuge tube, gently blow and beat
And it is mixed into individual cells suspension, uniform packing is into new culture bottle, and it is complete in each culture bottle to be separately added into 2mL
Culture solution is put into CO2Incubator carries out culture growth.
1.3 MTT experiments detect influence of the folic acid to PC12 cell Proliferation under lead exposure
1.3.1 MTT principle
MTT is a kind of crystalloid purple production that slightly solubility can be reduced by the succinate dehydrogenase in living cells mitochondria
The weld of object formazan, and this function can not be played in dead cell.Formazan is at specific solvent (such as DMSO)
It can be completely dissolved in the presence of in the case of, its absorbance, cell can be measured using instruments such as microplate reader under specific wavelength
The much faster then absorbance value (OD value) of proliferation is bigger, can reflect the quantity of living cells indirectly.
1.3.2 to the influence of PC12 cell proliferation rate
The preferable culture bottle of cell growing way after picking differentiation is added 1~2mL trypsase and comes when it covers with bottom of bottle 3/4
The PC12 cell for digesting bottom of bottle, it is 5 × 10 that cell concentration, which is made,5The individual cells suspension of/mL adds 100 μ l in the every hole of 96 orifice plates
Cell suspension, 200 μ l PBS, which are added, in each hole in culture plate edge prevents each hole culture solution in plate from volatilizing.Every group of each 8 hole,
Respectively with the PC12 cell of final concentration of 0,25,50,75,100 (μ g/mL), continue to put 37 DEG C, 5%C0 after liquid feeding2In incubator
Culture collect supernatant for 24 hours, when for 24 hours after, respectively from CO2One piece of 96 orifice plate is taken out in incubator, every hole is added before 20 μ l
Prepared MTT solution, is put into CO2After reacting 4h in incubator, 96 orifice plates are taken out, are placed under Biohazard Safety Equipment, it will be in hole
All liq discards, and notices that pipette tips not encounter each hole bottom of 96 orifice plates.Last every hole adds the DMSO of 150 μ l, by 96 holes
After being protected from light concussion 30 minutes in culture plate placement horizontal shaker, under enzyme-linked immunosorbent assay instrument, returned to zero using zeroing hole, simultaneously
Wavelength 490nm is set to read and record each group OD value.
2. experimental result is shown in Table 2, with Normal group cell survival rate (100%) for reference, the present invention is small containing sunflower disk
When the anti-gout composition of molecular peptide is acted on according to concentration (50 μ g/mL and 100 μ g/mL, 200 μ g/mL), apparent agent is shown
Dependence is measured, with the reduction of dosage, cell survival rate is gradually increased.It is computed, present invention small-molecular peptides containing sunflower disk resist
Cell survival rate is respectively reached in concentration 50 μ g/mL and 100 μ g/mL, 200 μ g/mL after the effect for 24 hours of gout composition
58.25% ± 0.05%, 47.35% ± 0.02%, 25.22% ± 0.01%.The result shows that present invention small molecule containing sunflower disk
The anti-gout composition of peptide has certain protective effect to PC12 cell.
Experimental result of the anti-gout composition of 2 small-molecular peptides containing sunflower disk of table to PC12 cell proliferation rate
| Drug concentration μ g/mL | Appreciation rate |
| 200 concentration | 25.22% |
| 100 concentration | 47.35% |
| 50 concentration | 58.25% |
Three, in the embodiment of the present invention 1 anti-gout composition of the small-molecular peptides containing sunflower disk to RAW264.7 macrophage
It influences
1. test method
The configuration of 1.1 reagents
Complete culture solution: 10% inactivated fetal bovine serum+90%DMEM high glucose medium, degerming, 4 DEG C of preservations.
Continual medium: 5% inactivated fetal bovine serum+95%DMEM high glucose medium, degerming, 4 DEG C of preservations.
MTT solution: 50mg MTT powder+10mL PBS, aseptic filtration ,~20 DEG C save backup.
Cells frozen storing liquid: inactivated fetal bovine serum and DMSO are uniformly mixed with 9:1.
The recovery of 1.2 RAW264.7 macrophages, is passed on and is frozen at culture
Recovery: after freeze-stored cell takes out 37 DEG C it is instant, take 15mL centrifuge tube, 10mL serum-containing media be added, will freeze
Liquid (usual 1~1.5mL) is added in above-mentioned centrifuge tube containing culture medium in pipe, and featheriness mixes, the centrifugation of 1200r/min room temperature
5min removes supernatant, and 3ml serum-containing media is added, and featheriness mixes to obtain cell suspension, is added in culture bottle, paving of rolling
It is flat.
Culture: cell is placed in adherent growth in the culture bottle containing complete medium, and condition of culture is 37 DEG C of constant temperature and 5%
CO2.80% or more of culture bottle bottom is covered with to cell, cell can be passed on.
Passage: abandoning culture solution in culture bottle, and appropriate trypsin solution digestion, which is added, disperses cell individually, about 1mL covering
Culture bottle bottom.Culture in right amount containing serum is added if cytoplasm retraction, space between cells increase in 37 DEG C of 2~5min of incubation immediately
Liquid terminates digestion.Bottle wall cell is gently blown and beaten to reiteration, adherent cell detachment bottle wall is made to suspend to form cell suspension.
Process is soft, prevents from overexerting and causes cellular damage.After blood counting chamber counts, it is connected to new culture bottle (one passes three), is put
Enter incubator culture.
It freezes: when cell is in logarithmic growth phase cell, cell suspension is made by above-mentioned propagating method, cell density is made to exist
5×107/ mL or so, centrifugation, removes supernatant.Cell is resuspended in cells frozen storing liquid, and movement is light as far as possible, is then sub-packed in sterile freeze
Pipe, 1mL/ pipe.Cryopreservation tube is sealed, is marked.Cryopreservation tube with 4 DEG C 10 minutes ,~20 DEG C 30 minutes ,~80 DEG C of overnight gradients are frozen
It deposits, finally throws in liquid nitrogen tank long term storage.
Influence of the anti-gout composition of 1.3 small-molecular peptides of the invention containing sunflower disk to RAW264.7 macrophage appreciation rate
For cell inoculation in 96 orifice plates, density is 2 × 105A/mL, 100 holes μ L/ of addition, incubator culture 2h, cell are complete
It is complete it is adherent after, replacement DMEM maintains culture solution culture.Normal group is that LPS and sample liquid is not added；LPS is added in culture solution in LPS group
Make its final concentration of 1 μ g/mL；The concentration gradient of 20~360 μ g/m L is respectively set in sample sets, each sample.It cultivates for 24 hours, every group
Do 5 multiple holes.Culture medium is outwelled later, and 20 hole μ L/ MTT solution, 37 DEG C of incubation 4h are added.It sucks and DMSO is added in hole after liquid
150 hole μ L/ of solvent, ELISA Plate are incubated for instrument and gently vibrate 10min, detect absorbance at 540nm later.
2. experimental result is shown in Table 3, with Normal group cell survival rate (100%) for reference, the present invention is small containing sunflower disk
When the anti-gout composition of molecular peptide is acted on according to concentration (50 μ g/mL and 100 μ g/mL, 200 μ g/mL), apparent agent is shown
Dependence is measured, with the increase of dosage, cell survival rate is gradually increased.It is computed, cell survival rate after the effect for 24 hours of Aodong compound
Respectively reach 15.2% ± 0.05% in concentration 50 μ g/mL and 100 μ g/mL, 200 μ g/mL, 16.2% ± 0.02%,
23.7% ± 0.01%.Show that the anti-gout composition of present invention small-molecular peptides containing sunflower disk has RAW264.7 macrophage
There is certain protective effect.
Experimental result of the anti-gout composition of 3 small-molecular peptides containing sunflower disk of table to RAW264.7 macrophage appreciation rate
| Drug concentration μ g/mL | Appreciation rate |
| 200 concentration | 23.7% |
| 100 concentration | 16.2% |
| 50 concentration | 15.2% |
In conclusion sunflower disk small-molecular peptides and Chinese medical extract prescription act in the present invention, there is synergy, it is right
Human umbilical vein endothelial cells, PC12 cell, RAW264.7 macrophage have proliferation function, show there is protective effect, explanation can
It is substantially reduced uric acid level, and there is anti-inflammatory, analgesic effect.In addition, sunflower disk, other a few herbs have dual-purpose of drug and food,
Feature without side-effects, synergistic effect is obvious, to treatment urarthritis significant effect, can take for a long time.During the present invention is
Medicine treatment gout provides effective method and theoretical foundation.
The synthesis result of the anti-gout composition In vitro cell experiment of 4 small-molecular peptides containing sunflower disk of table
Typical case
Curative effect of the invention is further demonstrated that by following typical case
Case 1
Lee, male 30 years old, routine physical examination detect uric acid level 532, drink the anti-of 1 small-molecular peptides containing sunflower disk of the embodiment of the present invention
After one wheat harvesting period of gout composition, uric acid level 431.1 is detected, uric acid decline 101 illustrates the small-molecular peptides of the invention containing sunflower disk
Anti-gout composition has the function of lower uric acid level, and significant effect.
Case 2
Wu, male 64 years old, routine physical examination detect uric acid level 494, have drunk 2 small-molecular peptides containing sunflower disk of the embodiment of the present invention
Anti-gout composition two months, check detection uric acid level 287 had been reduced to normal range (NR), and the gout at big toe position
Stone has been completely dissolved.
Case 3
Mr. Lin, male 29 years old gout 5 years, detect uric acid level 535, and gout is all broken out several times every year, has eaten the embodiment of the present invention
After the anti-gout composition of 1 small-molecular peptides containing sunflower disk, detection uric acid level is reduced to 263, and tophus starts the softening that becomes smaller.
Claims (10)
1. a kind of anti-gout composition of small-molecular peptides containing sunflower disk, which is characterized in that prepare the raw material and its again of the drug
It is as follows to measure number: 10~30 parts of sunflower disk, 20~60 parts of semen coicis, 20~60 parts of lophatherum gracile, 20~60 parts of rhizoma imperatae, Cortex Eucommiae are male
Spend 20~60 parts, 20~60 parts of hawthorn.
2. a kind of anti-gout composition of small-molecular peptides containing sunflower disk according to claim 1, which is characterized in that preparation institute
Raw material and its parts by weight for stating drug are as follows: 20 parts of sunflower disk, 40 parts of semen coicis, 40 parts of lophatherum gracile, 40 parts of rhizoma imperatae, Cortex Eucommiae
40 parts of male flower, 40 parts of hawthorn.
3. a kind of preparation method of the anti-gout composition of small-molecular peptides containing sunflower disk, which is characterized in that steps are as follows:
(1) parts by weight as described in claim 1 or 2 weigh each raw material；
(2) sunflower disk is taken to be soaked in water, flash extracter extracts 5~15 times, and extracting solution is centrifuged 10~30min, supernatant is taken,
Complex enzyme zymohydrolysis, is obtained by filtration the extracting solution that molecular weight is less than 10kd, and freeze-drying obtains sunflower disk small-molecular peptides freeze-dried powder；
(3) after taking semen coicis to soak in water, refluxing extraction obtains semen coicis extracting solution；
(4) after taking lophatherum gracile to soak in water, refluxing extraction obtains henon bamboo leaf extract；
(5) after taking rhizoma imperatae to soak in water, refluxing extraction obtains rhizoma imperatae extracting solution；
(6) after taking eucommia Bark male flower to soak in water, refluxing extraction obtains eucommia Bark male flower extracting solution；
(7) after taking hawthorn to soak in water, refluxing extraction obtains hawthorn extract；
(8) semen coicis extracting solution, henon bamboo leaf extract, rhizoma imperatae extracting solution, eucommia Bark male flower extracting solution and hawthorn extract are mixed
It closes, dried extract powder is obtained after spray drying；
(9) sunflower disk small-molecular peptides freeze-dried powder and dried extract powder are contained small point of sunflower disk after mixing up to described
The anti-gout composition of sub- peptide.
4. a kind of preparation method of the anti-gout composition of small-molecular peptides containing sunflower disk according to claim 3, feature
It is, the dosage of water described in step (2) is 10~20 times of sunflower disk quality.
5. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In with flash extracter extraction 5~15 times operations described in step (2), the time extracted every time is 2~10s.
6. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In complex enzyme described in step (2) is the mixed enzyme of pepsin and trypsase；
The temperature of the enzymatic hydrolysis is 35~37 DEG C, and enzymolysis time is 4~8h.
7. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In the temperature of freeze-drying described in step (2) is -40~50 DEG C, and pressure is 0.05~0.08kpa.
8. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In the additional amount of water described in step (3) is 10~20 times of semen coicis quality；
The additional amount of water described in step (4) is 10~20 times of henon bamboo leaf quality；
The additional amount of water described in step (5) is 10~20 times of rhizoma imperatae quality；
The additional amount of water described in step (6) is 10~20 times of eucommia Bark male flower quality；
The additional amount of water described in step (7) is 10~20 times of hawthorn quality.
9. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In the time of step (3) immersion described in either step into step (7) is to impregnate 0.5~1h.
10. according to a kind of preparation method of the anti-gout composition of the small-molecular peptides containing sunflower disk as claimed in claim 3, feature exists
In the operation of refluxing extraction described in step (3) to step (7) is with 1~3h of extractor refluxing extraction.
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| CN116173149A (en) * | 2023-03-24 | 2023-05-30 | 深圳万久投资发展有限公司 | Plant beverage capable of regulating metabolism of human body and preparation method thereof |
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Application publication date: 20191122 |