CN106699849A - Novel polypeptide and Periplaneta americana total polypeptide extract capable of promoting tissue repair, and application thereof - Google Patents
Novel polypeptide and Periplaneta americana total polypeptide extract capable of promoting tissue repair, and application thereof Download PDFInfo
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Abstract
The invention specifically relates to novel polypeptide and Periplaneta americana total polypeptide extract capable of promoting tissue repair, and application thereof, belonging to the fields of traditional Chinese medicines and crude drugs. The novel polypeptide capable of promoting tissue repair is named as Periplapeptide A and has a structure as shown in a formula I H-Ala-Ala-Pro-Tyr-Ala-His-Val-Ala-Ala-Pro-Tyr-Ala-His-Phe-OH. The Periplaneta americana total polypeptide extract capable of promoting tissue repair is prepared by subjecting the bodies of Periplaneta americana to alcohol extraction, degreasing and enrichment via a reversed-phase material, wherein the mass percentage content of total polypeptide is greater than 50%, and the extract contains Periplapeptide A. The novel polypeptide and the Periplaneta americana total polypeptide extract containing novel polypeptide in the invention can promote propagation of mouse embryo fibroblasts and migration of immortalized human epidermal cells at low concentrations, have low toxic and side effect and good medicinal prospects, and are applicable to preparation of medicines used for treating wounds, burns and ulcers or cosmetics and daily chemical articles used for beautifying the skin.
Description
Technical field
The present invention relates to Chinese medicine and natural medicine field, and in particular to a kind of novel polypeptide that promotion organization is repaired and
The total polypeptide extract of American cockroach and application.
Background technology
Agglutination after tissue injury is the process of various repairing cell proliferations, differentiation, migration and apoptosis.
Various acute and chronic injuries such as inflammation, ulcer, burn, wound, operation and congenital abnormality can cause big face
Product defect of skin, dermal chronic ulcer and scar, and trigger the reaction of many parts and whole body system, its harm
It is extremely serious.Polypeptide drugs have uniqueness in terms of wound healing, prevention of scar and treatment chronic ulcer
Advantage.The polypeptides such as its Concentrations of Epidermal Growth Factor (hEGF), basic fibroblast growth factor (bFGF)
With obvious reparation wound, skin care, crease-resistant and anti-aging effect, but their preparation cost is high, stable
Property it is poor, limit its application.
American cockroach (Periplaneta americana L.) is Arthropoda Insecta Blattaria Blattidae
(Blattidae) Periplaneta (Peripalneta) insect, is commonly called as cockroach, widely distributed in China.As one
Traditional Chinese medicine is planted, American cockroach has long history of being used as medicine, is recorded in earliest《Sheng Nong's herbal classic》, referred to as
Roach is honest and clean, for treating the diseases such as sore and toxic, infantile malnutrition.Modern pharmacology research shows that American cockroach has
There are obvious promotion organization reparative regeneration, antitumor and antihepatitic activity (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2007,32:
2326-2331).At present, the marketed drug being made as primary raw material with American cockroach has Kangfuxin Liquid, heart arteries and veins
Grand capsule, Ganglong capsule etc., especially Kangfuxin Liquid are at aspects such as treatment burn, scald and digestive tract ulcers
With good efficacy, but its active ingredient is unclear.
Existing document report American cockroach ethanol extract has promotion cell migration, and (the Zhejiang combination of Chinese tradiational and Western medicine is miscellaneous
Will, 2012,9:689-691) and promote cell migration effect (Chinese medicine with it is clinical, 2013,1:34-36), but not
Specify its active principle or active ingredient；Chinese invention patent 200710067707.4 describes the reversed material system of using
The method of standby American cockroach antitumor active site, but not yet explicitly chemical composition type and it is not related to promotion organization
The effect of reparation；Chinese invention patent ZL201210137422.4 discloses a kind of system of cockroach polypeptide class material
Preparation Method and its anti-herpesvirus medical usage, Chinese invention patent ZL201210137410.1 describe one kind
The preparation of cockroach polypeptide and its resisting gram-positive and negative bacterium medical usage, but above-mentioned patent do not illustrate not only
The specific chemical composition at polypeptide position, is also not directed to total polypeptide and polypeptide monomer in terms of promotion organization reparation
Using.
The content of the invention
In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the invention is to provide a kind of promotion group
Knit the novel polypeptide of reparation.
Another object of the present invention is to provide the total polypeptide extract of American cockroach that a kind of promotion organization is repaired, should
Total polypeptide contains above-mentioned novel polypeptide composition.
Another object of the present invention is to provide novel polypeptide and the total polypeptide of American cockroach that above-mentioned promotion organization is repaired
The preparation method of extract.
It is still another object of the present invention to provide above-mentioned novel polypeptide and the total polypeptide extract of American cockroach in promotion group
Knit the application in terms of reparation.
The purpose of the present invention is achieved through the following technical solutions：
The novel polypeptide that a kind of promotion organization is repaired, is named as Periplapeptide A, and its complete sequence primary structure is such as
Shown in Formulas I：
H-Ala-Ala-Pro-Tyr-Ala-His-Val-Ala-Ala-Pro-Tyr-Ala-His-Phe-OH
Formulas I；
A kind of total polypeptide extract of American cockroach that promotion organization is repaired, total polypeptide extract contains above-mentioned new
Polypeptide Periplapeptide A, and in extract total polypeptide weight/mass percentage composition ＞ 50%；
The preparation method of the total polypeptide extract of American cockroach that described promotion organization is repaired, comprises the steps of：
The American cockroach polypide crushed or do not crush is extracted with hydrous ethanol, extract concentration adds 10 times of bodies
Accumulated heat water (70 DEG C), is incubated stratification, upper strata fluid is discarded, through anti-phase C-18 posts after aqueous solution concentration
Chromatogram, tracing detection is analyzed using liquid chromatography-mass spectrography (HPLC-MS) method, is collected and is contained polypeptide compound
Stream part, merge stream part, be concentrated under reduced pressure or be vacuum dried, the American cockroach for being promoted tissue repair is always more
Peptide extract；
The preparation method of the novel polypeptide that described promotion organization is repaired, comprises the steps of：
The total polypeptide extract of American cockroach that above-mentioned promotion organization is repaired is further by reverse phase silica gel post layer
Analysis, Sephadex G-50 column chromatographies and Reverse phase preparative HPLC are isolated and purified, and are promoted tissue repair
Novel polypeptide Periplapeptide A；
The novel polypeptide Periplapeptide A or the total polypeptide extract of American cockroach that described promotion organization is repaired exist
Prepare the application in the product of promotion organization gentrify skin aesthetic effect；
Described product is preferably medicine, cosmetics or daily chemical product；
It is further preferred that the novel polypeptide or the total polypeptide extract of American cockroach of described promotion organization reparation exist
Preparation control treatment trauma, burn, the medicine of ulcer or improve skin aesthetic feeling cosmetics and daily chemical product in answer
With；
Described medicine, cosmetics or the Periplapeptide A containing effective dose or America in daily chemical product
The total polypeptide extracts of big Lian, balance of auxiliary material or other compatible medicines；
Described auxiliary material refers to conventional excipient, such as solvent, disintegrant, flavouring, preservative, coloring
Agent and adhesive etc.；
The compatible medicine of other described, refers to other natural drugs or chemicals；
Described medicine, cosmetics or daily chemical product can use tablet, capsule, injection, liposome
Nanoparticle, controlled release agent, gel ointment, ointment, externally-applied liniment, patch (such as facial mask), creme (example
Such as face cream), detergent (such as shampoo, perfumed soap, bubble bath or bath salt), emulsion, gel, toner；
The present invention has the following advantages and effect relative to prior art：
(1) present invention is separated from American cockroach and identified with the new many of promotion organization repair first
Peptide compounds Periplapeptide A, complete sequence primary structure is shown in formula I.
(2) the total polypeptide extract of American cockroach of the invention includes above-mentioned novel polypeptide Periplapeptide A, and
Total content of peptides is more than 50%.
(3) novel polypeptide of the invention and the total polypeptide extract of the American cockroach containing the compound are in low concentration
The propagation of MEC and people can be promoted to immortalize epidermis under (0.39~3.13 μ g/mL) thin
The migration of born of the same parents.
(4) novel polypeptide Periplapeptide A of the invention and the total polypeptide of the American cockroach containing the compound are carried
Take thing toxicity in vivo very low, do not observe obvious toxic and side effect under oral 400mg/kg dosage.
(5) novel polypeptide Periplapeptide A of the invention and the total polypeptide of the American cockroach containing the compound are carried
Thing good stability is taken, it is orally available, it is difficult to be hydrolyzed by hydrochloric acid in gastric juice etc., can be used for tissue repair and treatment alimentary canal is burst
Ulcer.
(6) novel polypeptide of the invention and the total polypeptide extract of the American cockroach containing the compound have whitening,
Go wrinkle, scar and color spot to act on, can obviously improve the skin aesthetic feeling of subject.
Brief description of the drawings
Fig. 1 is polypeptide Periplapeptide A1H NMR spectras.
Fig. 2 is polypeptide Periplapeptide A13C NMR spectras.
Fig. 3 is the amino acid sequencing spectrogram of polypeptide Periplapeptide A.
Fig. 4 is interpretation of result figure of the total polypeptide extract of American cockroach to Balb/c 3T3 cell proliferative effects.
Fig. 5 is interpretation of result figures of the polypeptide Periplapeptide A to Balb/c 3T3 cell proliferative effects.
Fig. 6 is the interpretation of result figure that the total polypeptide extract of American cockroach influences on Hacat cell migrations.
Fig. 7 is the interpretation of result figure that polypeptide Periplapeptide A influence on Hacat cell migrations.
Fig. 8 is the system that the total polypeptide extract of American cockroach and Periplapeptide A influence on Hacat cell migrations
Meter interpretation of result figure.
Fig. 9 is the HPLC-MS chromatograms of the total polypeptide extract of American cockroach.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiment party of the invention
Formula not limited to this.
The condition of liquid chromatography-mass spectrography (HPLC-MS) analysis that implementation below is used is as follows：
Laboratory apparatus is Agilent 6210LC/MSD TOF LC-MS instrument, mass range：100-3000,
Drainage pattern：Positive ion mode, Gas Temp：300 DEG C, Dring Gas：11L/min, Nebulizer：
35psig, Vcap：2000V, Fragmentor：120V, Skimmer：65V.Analytical column is YMC C8
Chromatographic column (4.6 × 250mm, 5 μm), chromatographic condition：Mobile phase A (the formic acid of volume fraction 0.1%
Water), Mobile phase B (acetonitrile), 0min (A:B=95:5) to 90min (A:B=10:90) gradient is washed
It is de-.
The preparation of the total polypeptide extract of the American cockroach of embodiment 1
American cockroach medicinal material 5kg, is ground into meal, is extracted with 3 times the 80% of quality (v/v) ethanol percolations
3 times, every time 24 hours.Merge extract solution, be concentrated under reduced pressure into thick paste about 1.5L, add 10 times of volume heat
Water (70 DEG C), insulation stands 12 hours, layering, discards upper strata fluid.Loading after lower aqueous solution concentration
It is first that 30% methyl alcohol elutes 6 column volumes with percent by volume, then use body respectively in anti-phase C-18 chromatographic columns
Product percentage is that 50% and 75% methanol aqueous solution elutes 6 column volumes, and tracking inspection is analyzed with HPLC-MS
Survey, collect the main stream part containing polypeptide compound, merge stream part, be concentrated under reduced pressure or be vacuum dried, obtain U.S.
The total polypeptide extracts of the big Lian in continent (56g) (Fig. 9).
The separation of the polypeptide Periplapeptide A of embodiment 2 and Structural Identification
(1) taking the total polypeptide extract 20g of American cockroach carries out reverse phase silica gel (ODS) column chromatography, with methyl alcohol
- water is eluant, eluent, is 10 according to methyl alcohol and water volume ratio:90、20:80、30:70、50:50、70:30、90:10
Gradient eluted, obtain 6 main flows part Fr.A~Fr.F；
(2) Sephadex G-50 chromatograms are splined on to the stream part Fr.C (2.5g) obtained in step (1)
Post, with water as mobile phase, flow velocity is eluted for 0.5mL/min, is analyzed through HPLC-MS and detected, is closed
And similar stream part is collected, obtain 5 Arius part Fr.C1~Fr.C5；
(3) dissolved with the methyl alcohol of volume fraction 5% after step (2) being obtained into Fr.C4 concentrations, then with anti-
Phase preparation HPLC is isolated and purified, and is 40 with volume ratio:60 acetonitrile-water is eluant, eluent, and flow velocity is 3
ML/min is eluted, and collects the chromatographic peak of retention time 30.8min, obtains Periplapeptide A (21.8
mg)。
(4) structural characterization of Periplapeptide A
White powder；UV(MeOH)λmax(logε)206(3.99),278(2.88)nm；IR(KBr)νmax
3400,2924,2854,1650,1434,1386,1118,1072,471cm-1；HR-ESI-MS m/z
1485.7276[M+H]+(C72H96N18O17, calculated value 1485.7274).Hydrogen spectrum (1H NMR) and carbon spectrum
(13C NMR) data are shown in Table 1 (being belonged to by 2D-NMR).1H,13C NMR spectras and amino acid sequencing
Result is shown in Fig. 1~3.According to above physics and chemistry and spectroscopic data, the chemical constitution of Periplapeptide A is identified,
As shown in formula I.
The Periplapeptide A's of table 11H (500MHz) and13C (125MHz) NMR data
Note：(DMSO-d6,, δ units are ppm, and J units are Hz, and overlapped signal does not mark peak shape)
The synthesis of the novel polypeptide Periplapeptide A of embodiment 3
Using full-automatic polypeptide synthetic instrument conventional solid synthetic method, by resin swelling, deprotection, washing,
The processes such as amino acid dissolving, activation of amino acid, condensation, complete the synthesis of novel polypeptide Periplapeptide A,
Obtain product 5.0g.
The assay of polypeptide in the total polypeptide extract of the American cockroach of embodiment 4
(1) biuret colorimetric method
Take 12 test tubes and be divided to two groups, be separately added into 0,0.2,0.4,0.6,0.8,1.0 milliliter of standard protein
Matter solution (crystallization bovine serum albumin, 10mg/mL), is supplied to 1 milliliter with water, is subsequently adding 4 milliliters
Biuret reagent.After fully shaking up, placed 30 minutes under room temperature (20~25 DEG C), at 540nm
Carry out colorimetric estimation.With not plus protein solution first test tube as blank liquid.Take two groups of measure
Average value, the content with protein as abscissa, absorbance value be ordinate draw standard curve.
The aqueous solution 1mL of determinand (the total polypeptide extract of American cockroach of the present invention) is added in test tube, so
4mL biuret reagents are added afterwards.Reacted by above-mentioned condition, trap is determined under 540nm.
Measurement result shows, the weight/mass percentage composition ＞ 50% of polypeptide in the total polypeptide extract of American cockroach.
(2) Coomassie brilliant blue colorimetric method
Take 12 test tubes and be divided to two groups, the concentration for being separately added into different volumes is 100ug/mL bovine serum albumins
Titer, replenishes water to 1.0mL.Blank cuvette adds 1.0mL distilled water.Then every test tube adds 5.0
ML Coomassie brilliant G-250 reagents, shake up placement 5min (20~25 DEG C), in UV-vis spectroscopy light
Light absorption value is determined at degree meter 595nm.With A595 light absorption values as ordinate, the content of bovine serum albumin is
Abscissa draws standard curve.
The aqueous solution 1mL of determinand (the total polypeptide of American cockroach of the present invention) is added in test tube, is subsequently adding
5mL Mas bright blue G-250 reagents.Reacted by above-mentioned condition, trap is determined under 595nm.
Measurement result shows, the weight/mass percentage composition ＞ 50% of polypeptide in the total polypeptide extract of American cockroach.
The polypeptide Periplapeptide A of embodiment 5 and the total polypeptide extract of American cockroach are thin to Balb/c 3T3
The facilitation of born of the same parents' propagation
By cell (the Balb/c 3T3 cell lines, purchased from American Type Culture collection warehousing in exponential phase
American Type Culture Collection, ATCC) with pancreatin digest, centrifugation, it is resuspended after carry out cytometer
Number, is inoculated in 96 orifice plates by 3000 cells/wells, is patted to cell and is uniformly dispersed, PBS closings.37
DEG C, 5%CO2Overnight incubation.Culture medium is abandoned, the starvation media treatment 24h containing 0.5%FBS is added,
Make cell synchronization.Starvation media is abandoned, adds complete medium to configure the polypeptide of various concentrations
Periplapeptide A and the total polypeptide extract of American cockroach, 37 DEG C, 5%CO2Incubator is incubated 48h.Press
10 μ L/ holes add CCK8 reagents, 37 DEG C of 1~4h of incubation, the inspection of ELIASA 450nm, 630nm dual wavelength
Survey.Calculate cell survival rate.Repeat experiment at least more than 3 times.
As shown in Figures 4 and 5, Periplapeptide A are at low concentration (0.39~3.13 μ g/mL) for result of the test
There is significant proliferation to Balb/c 3T3 cells, and its activity to be significantly stronger than marketed drugs rehabilitation new
Liquid (Fig. 5)；The total polypeptide extract of American cockroach has when less than 6.25 μ g/mL to Balb/c 3T3 cells
Stronger proliferation, and its activity is better than marketed drugs Kangfuxin Liquid (Fig. 4).
The polypeptide Periplapeptide A of embodiment 6 and the total polypeptide extract of American cockroach are moved to Hacat cells
The facilitation of shifting
The HaCAT cells of exponential phase (are purchased from American Type Culture collection warehousing American Type
Culture Collection, ATCC), pancreatin digestion is resuspended, by 1 × 106Individual/hole is inoculated into 12 orifice plates, puts
37 DEG C, 5%CO2Culture to cell covers with culture hole in incubator.Nutrient solution, PBS washings, with nothing are abandoned in suction
Bacterium pipette tips are rule from top to bottom in each test hole central vertical in culture hole.Wash away what cut was produced with PBS
Cell mass, makes scratching edge neat.Add fresh culture and the total polypeptide extract (1.56 of American cockroach
μ g/mL) or Periplapeptide A (1.56 μ g/mL), taken pictures under microscope, continue to be placed in cell culture incubator
Middle culture.Every 12 hour observed and recordeds, gained view data is processed with Image Pro Plus 6.0.
Cut uniformly chooses 30 points per lateral edges, takes its center line and represents scratching edge, measures cut spacing, is used in combination
Below equation calculates scratch removal rate：Scratch removal rate=(0h scratch widths-different time points scratch width)
/ 0h scratch widths.
Result of the test shows that the mobility of Periplapeptide A group HaCAT cells is up to 87% (72 hours)
(Fig. 7, Fig. 8), the mobility of the total polypeptide group HaCAT cells of American cockroach reaches 79% (72 hours) (figure
6, Fig. 8), obviously higher than control group 38% (72 hours) (Fig. 8), polypeptide Periplapeptide A are illustrated
With the lateral transfer that the total polypeptide extract of American cockroach can be obviously promoted HaCAT cells.
The polypeptide Periplapeptide A of embodiment 7 and the total polypeptide extract of American cockroach are damaged to mouse skin
Repair
40 8~10 weeks male mouse of kunming (being purchased from Guangdong Province's animal center) sub-cage rearing is chosen, is freely taken
Food, experiment the previous day stops feed.It is small with the yellow Jackets 0.2mL intraperitoneal injection of anesthesia of mass fraction 1%
Mouse, back cropping, backbone epidermal skin is symmetrical above and below to open 0.5cm × 0.5cm square wounds, cuts off complete
Layer skin, does not injure muscle layer, and wound hemostasis are standby.Next day, etherization mouse, wound is added dropwise different
The total polypeptide extract of the Periplapeptide A of solubility, American cockroach, physiological saline and each 0.1mL of Kangfuxin Liquid,
Daily dressing is once.Wound healing situation was observed in 3,5,10 days.Surface of a wound area is first depicted in thin transparent
On film, then as template, homogeneous hard paper is cut into onesize, is then claimed with assay balance
Quality.Surface of a wound size is indirectly indicative with cardboard tablet quality.Wound healing rate is calculated by formula：The surface of a wound
Healing rate (%)=(original surface of a wound area-do not heal surface of a wound area)/original surface of a wound area.The results are shown in Table 2.
The wounds in mice healing rate statistics of table 2
Note：Compared with model group, * P ＜ 0.05 have significant difference
Result of the test shows that polypeptide Periplapeptide A and the total polypeptide extract of American cockroach can substantially promote
Enter the healing of mouse skin wound, compare with significant difference with control group, and better than Kangfuxin Liquid.
The protection of embodiment 8Periplapeptide A and the total polypeptide of American cockroach to stress in rats gastric ulcer is made
With
Test method：180~210g of body weight male SD rats 50 (purchased from Guangdong Province's animal center) are taken,
It is randomly divided into blank group, model group, ulcerlmin group (1g/kg, positive drug group), low dose group and high dose
Group.Administration group gastric infusion, continuous 7 days.Water is can't help in 24h fasting before modeling, in addition to control group, is used
Constraint water seaoning modeling, mouse plate is fixed on by each group rat, and head is vertically immersed in constant temperature (20 DEG C) water upwards
8h in groove, the water surface is flushed with rat xiphoid-process.Rat cervical dislocation stress be put to death after modeling terminates, cut open the belly,
Ligation pylorus.To the formalin 2mL of intragastric infusion volume fraction 10%, orifice of the stomach is ligatured, take out body of stomach and be placed in
15min is fixed in formalin.Cut off along greater curvature, launched after normal saline flushing, observation stomach lining is damaged
Hinder and calculate ulcer index.By Guth criterion calculations UI (UI)：Petechial hemorrhage is calculated as 1 point,
Streak-like hemorrhage length<1mm is 2 points, and 1~2mm is 3 points, and 2~4mm is 4 points,>4mm is 5
Point, width>Score value × 2 during 1mm.The results are shown in Table 3.
The rat ulcer index statistics of table 3
Note：Compared with model group, * P ＜ 0.01 have significant difference
Result of the test shows that polypeptide Periplapeptide A and the total polypeptide extract of American cockroach are to stress in rats
Gastric ulcer has obvious protective function, compares with model group with significant difference, and its protecting effect is better than
Positive drug ulcerlmin.
The acute toxicity testing of the polypeptide Periplapeptide A of embodiment 9 and the total polypeptide extract of American cockroach
Test method：Take the Kunming mouse (purchased from Guangdong Province's animal center) of 18~22g of body weight, random point
Group, every group 10, the polypeptide Periplapeptide A of gavage various dose or the total polypeptide extract of American cockroach,
According to mouse survival situation, half lethal dose LD is calculated50.Median lethal dose is calculated as follows：Half
Number lethal dose (mg/kg)=(body weight of dosage/correspondence mouse when mouse half is dead).
Result of the test：Polypeptide Periplapeptide A and the total polypeptide extract of American cockroach are 100~400
Under the dosage of mg/kg, dead mouse is not observed, illustrate that their toxicity is very low, LD50It is more than
400mg/kg (table 4).
The Acute Toxicity of the Periplapeptide A of table 4 and the total polypeptide extract of American cockroach to mouse
The cutaneous safety of the polypeptide Periplapeptide A of embodiment 10 and the total polypeptide extract of American cockroach is commented
Valency
Tested crowd：Between 18~50 years old age, the people of women 38, the people of male 12 amounts to 50 people.Receive
Examination person's skin health, without skin disease allergies, meets subject's aspiration inclusion criteria.
Test method：From qualified patch device, in closed patch test method, by polypeptide Periplapeptide
A or the total polypeptide extract of American cockroach about 0.020~0.025mL (2mg/mL) are dripped in patch device, outward
Be covered on subject back with special adhesive tape, trial target removed after 24 hours, respectively at 0.5 after removal, 6,
12nd, observation dermoreaction in 24,48 hours, according to《Cosmetics health specification》Middle dermoreaction grade scale
Record its result.
Result of the test：50 subjects of this experiment are small 0.5,6,12,24,48 by patch test
When observe dermoreaction, do not observe skin adverse reaction, illustrate polypeptide Periplapeptide A of the invention
Safety is used to skin with American cockroach total polypeptide extract.
The preparation of the tablet of embodiment 11
Polypeptide Periplapeptide A 0.1g, lactose 40g, appropriate starch slurry, magnesium stearate 0.2g, mixing,
Sieving, compressing tablet after drying.The every 0.001g of A containing Periplapeptide.
The total polypeptide extract 1g of American cockroach, preparation method is ibid.Every total polypeptide extract containing American cockroach
0.01g。
The preparation of the injection of embodiment 12
Polypeptide Periplapeptide A 0.1g, propane diols 50g, grinding, then add a small amount of water for injection dilution,
Mix, it is appropriate to be subsequently adding sodium chloride, and water for injection to 1000mL is added after dissolving, adjust pH value 5.5~
6.5, filtration, embedding, sterilizing obtains final product 1000 injection injections.
The total polypeptide extract 1g of American cockroach, preparation method ibid, obtains 1000 injection injections.
The preparation of the solid lipid nano-particles of embodiment 13
Polypeptide Periplapeptide A 0.1g, soybean lecithin 500mg, are dissolved in 25mL ethanol, separately take
Stearic acid 200mg and soybean lecithin 500mg are dissolved in 25mL hexamethylenes, mixing and stirring.In
The rotary evaporation that depressurized in 37 DEG C of waters bath with thermostatic control removes organic solvent, medicine and auxiliary material is formed uniformly in flask walls
Lipid membrane, stands overnight in vacuum desiccator, eliminates organic solvent；Another taking polyethylene glycol monostearate
Ester 3750mg, stirring and dissolving is added in above-mentioned film in 175mL water, and ultrasonic 10min is settled to
250mL, obtains light yellow transparent solution.This solution freeze-drying can be obtained into freeze-dried powder.With ball mill grinding 24
Hour, the nanoparticle of uniform particle sizes is obtained, mix and dispense.Every bag of 0.001g of A containing Periplapeptide.
The total polypeptide extract 1g of American cockroach, preparation method ibid, every bag of total polypeptide extract containing American cockroach
0.01g。
The preparation of the controlled release capsule of embodiment 14
Polypeptide Periplapeptide A 0.1g, lactose 40g and starch slurry be directly attached in right amount rotating pelletizer/
It is coated device and prepares particle, the plasticized ethylcellulose coating agent suspension spray of 15% solid to U.S. will be diluted to
On the revolving bed of the continent total polypeptide particles of big Lian.During spraying, the dispersion being made of PLURONICS F87 is carried
Body film coating particle, forms the particle that average particle size is about 450 μm of sustained release.Mix and load glue
Capsule, each capsule A containing Periplapeptide 0.001g.
The total polypeptide extract 1g of American cockroach, ibid, each capsule total polypeptide containing American cockroach is carried preparation method
Take thing 0.01g.
The preparation of the external-use gel paste of embodiment 15
Glycerine 2g is weighed, polyacrylic acid 0.75g and aluminium hydroxide 0.1g is sequentially added, is sufficiently mixed, added
1. the total polypeptide extract 2g of American cockroach, be thoroughly mixed under vacuum；Purified water is weighed in addition
5g, lactic acid 0.06g, sodium carboxymethylcellulose 0.1g are dissolved in water and obtained 2.；In being 2. added to 1.,
It is sufficiently stirred under vacuum condition, crosslinked reaction obtains ointment-containing body, by ointment-containing body coating (control pastille
Paste layer thickness is in 1.0mm or so), the specification of conventional emplastrum is cut into, the amount for often pasting drug containing is 0.2
Gram, dry, pack.
The preparation of the ointment of embodiment 16
Stearyl alcohol 20g melts in a water bath with albolene 21g, is heated to 75 DEG C, standby.Dodecyl
Sodium sulphate 1.46g, methyl hydroxybenzoate 0.025g, Nipasol 0.015g, propane diols 10g, American cockroach is total
Polypeptide extract 2g is soluble in water successively, is heated to 75 DEG C, and 75 DEG C of stearyl alcohols and albolene are added,
Stirring is made the ointment containing the total polypeptide extract 2% of American cockroach to cold.
The preparation of the externally-applied liniment of embodiment 17
The total polypeptide extract 1g of American cockroach is taken, is dissolved in 45mL distilled water, filtered, filtrate adds sweet
Oily 5mL, azone 1mL, distilled water supplies volume to 50mL, is made containing the total polypeptide extract of American cockroach
2% externally-applied liniment.
The preparation of the face cream of embodiment 18
By No. 26 white oil 1g, cetostearyl alcohol 1g, stearic acid 1g, monoglyceride 0.5g, 350 silicone oil 0.05
G, GTCC 0.5g, methyl hydroxybenzoate 0.03g mix, and are heated to 90 DEG C, are made the first semi-finished product；
Added in first semi-finished product deionized water 4g, the total polypeptide extract 1g of American cockroach, peregal -200.3g,
Glycerine 0.5g, and it is heated to 80 DEG C, the second semi-finished product of formation；Second semi-finished product are carried out 2 times at 80 DEG C
Homogeneous (3000 revs/min of rotating speed, 10 minutes time) continues stirring 30 minutes afterwards, is cooled to 45 DEG C
0.005g Kathon CGs are added after forming cream shape, is mixed, obtain the face containing the total polypeptide extract of 10% American cockroach
Frost.
The preparation of the facial mask of embodiment 19
The total polypeptide extract of 1g American cockroaches is dissolved in 10mL deionized waters, is filtered, filtrate and glycerine
The Carbomer 0.3g mixing of wetting, adds triethanolamine, and regulation pH value is 6~7, is spread evenly across facial mask
Paper, is made every facial mask containing the total polypeptide extract 0.1g of American cockroach.
0.1g Periplapeptide A are dissolved in 10mL deionized waters, remaining preparation method ibid, is made
Into every facial mask containing Periplapeptide A 0.01g.
The preparation of the toner of embodiment 20
The total polypeptide extract of 1g American cockroaches is configured to the aqueous solution of 2mg/mL；Glycerine 5g is added saturating
Disperse in bright matter acid 1g, be dissolved in water, stir, obtain hyaluronic acid solution；By trehalose 1g and urine
Mix with above-mentioned hyaluronic acid solution after Bursin 1g water dissolves, stir, obtain mixed solution；By America
The total polypeptide solutions of big Lian, D-pantothenyl aleohol 1g, avenabeta glucosan 10g, 1,2- pentanediol 10g and preservative
0.05g sequentially adds above-mentioned mixed solution, adds water and stirs, and obtains final product toner.
The preparation of the emulsion of embodiment 21
(1) the total polypeptide extract of 1g American cockroaches is configured to the aqueous solution of 2mg/mL；
(2) 0.05g Sodium Hyaluronates are dissolved in water dispersion, are stirred, obtain hyaluronic acid solution；
(3) by glycerine 5g, butanediol 3g, trehalose 2g, allantoin 0.2g, thickener 0.1g,
With water dissolves, 75 DEG C are heated to；
(4) by jojoba seed oil 2g, Nexbase 2004 3g, dimethyl silicone polymer 2g, the octanoic acid/last of the ten Heavenly stems
Sour glyceryl ester 3g, emulsifying agent 3g are heated to 75 DEG C, stir；
(5) product obtained in step (3) is quickly poured into product obtained in step (4), constant temperature is equal
3~5min of matter, cooling；
(6) less than 60 DEG C are cooled to, (1), (2), homogeneous are added；Less than 40 DEG C are cooled to, are added
Preservative and essence, obtain final product emulsion.
The preparation of the face cleaning gel of embodiment 22
The total polypeptide extract of 1g American cockroaches is configured to the aqueous solution of 2mg/mL；By above-mentioned American cockroach
Total polypeptide solution, glycerine 2.5g, Plantacare 818 3g, cocounut oil acyl apple amino acid sodium 3g, laruyl alcohol gather
Ether sodium sulfovinate 1.5g, solubilizer 0.1g, essence 0.05g are stirred in sequentially adding water, add thickener
0.5g, stirring to complete swelling can obtain face cleaning gel.
0.1g Periplapeptide A are configured to the aqueous solution of 0.2mg/mL, remaining preparation method ibid,
Obtain final product face cleaning gel.
The cosmetic result evaluation of the total polypeptide emulsion of the American cockroach of the present invention of embodiment 23
1.1 trial targets：The total polypeptide extract emulsion of American cockroach prepared by the embodiment of the present invention 21
1.2 tested crowds：Between 18~55 years old age, the people of women 40, the people of male 10 amounts to 50 people.
Subjects skin has wrinkle, and color spot, color and luster is low and deep, and minimally invasive, post-surgical scars etc. are left on skin no
Aesthetic considerations.
1.3 method of testings：After subjects skin's cleaning, on lotion applicator prepared by embodiment 21 to skin,
Observe and experience using effect.
1.4 test assessment results are as follows：
(8 weeks) on probation feedback conclusive tables of the total polypeptide extract emulsion of the American cockroach of table 5
Subject represents, using the total polypeptide product emulsion of American cockroach, can improve skin water content, strengthens flesh
Skin moisturizing power and skin elasticity, improve the tender moist sense of skin water.The product has whitening, removes wrinkle, scar
With the effect of color spot.There is not allergic phenomena in subject.
The cosmetic result evaluation of the Periplapeptide A facial masks of the present invention of embodiment 24
1.1 trial targets：Periplapeptide A polypeptide masks prepared by the embodiment of the present invention 19
1.2 tested crowds：Between 18~55 years old age, the people of women 41, the people of male 9 amounts to 50 people.
Subject's facial skin has wrinkle, and color spot, color and luster is low and deep, and minimally invasive, post-surgical scars are left on skin
Deng unsightly factor.
1.3 method of testings：After subject face cleaning, Periplapeptide A faces prepared by embodiment 19
Film is coated on face, once a day, observes and experience using effect.
1.4 test assessment results are as follows：
(8 weeks) on probation feedback conclusive tables of the American cockroach Periplapeptide A polypeptide masks of table 6
Subject represents, using Periplapeptide A polypeptide mask products, can improve skin water content, increases
Strong skin moisturizing power and skin elasticity, improve the tender moist sense of skin water.Product has whitening, goes wrinkle, scar
The effect of trace and color spot.There is not allergic phenomena in subject.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention do not receive above-mentioned reality
Apply the limitation of example, it is other it is any without departing from the change made under Spirit Essence of the invention and principle, modification,
Substitute, combine, simplify, should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (9)
1. the novel polypeptide that a kind of promotion organization is repaired, it is characterised in that：With structure shown in Formulas I, it is named as
Periplapeptide A；
H-Ala-Ala-Pro-Tyr-Ala-His-Val-Ala-Ala-Pro-Tyr-Ala-His-Phe-OH
Formulas I.
2. the total polypeptide extract of American cockroach that a kind of promotion organization is repaired, it is characterised in that：Wanted containing having the right
Seek the Periplapeptide A described in 1, and wherein total polypeptide weight/mass percentage composition>50%.
3. the preparation method of the total polypeptide extract of American cockroach that promotion organization described in claim 2 is repaired,
It is characterized in that comprising the steps of：
The American cockroach polypide crushed or do not crush is extracted with hydrous ethanol, extract concentration adds 10 times of bodies
Accumulated heat water, is incubated stratification, discards upper strata fluid, through anti-phase C-18 column chromatographys after aqueous solution concentration, adopts
Tracing detection is analyzed with liquid chromatography-mass spectrography method, the stream part containing polypeptide compound is collected, merges stream part, subtracted
Pressure concentration is vacuum dried, and is promoted the total polypeptide extract of American cockroach of tissue repair.
4. the preparation method of the novel polypeptide that promotion organization described in claim 1 is repaired, it is characterised in that include
Following steps：
The total polypeptide extract of American cockroach that claim 3 is prepared is further by reverse phase silica gel post layer
Analysis, Sephadex G-50 column chromatographies and Reverse phase preparative HPLC are isolated and purified, and are promoted tissue repair
Novel polypeptide Periplapeptide A.
5. the promotion organization described in claim 1 or 2 is repaired novel polypeptide or the total polypeptide extract of American cockroach
Application in the product for preparing promotion organization gentrify skin aesthetic effect.
6. promotion organization according to claim 5 is repaired novel polypeptide or the total polypeptide extract of American cockroach
Application in the product for preparing with tissue repair or improving skin aesthetic effect, it is characterised in that：
Described product is medicine, cosmetics or daily chemical product.
7. promotion organization according to claim 6 is repaired novel polypeptide or the total polypeptide extract of American cockroach
Application in the product for preparing with tissue repair or improving skin aesthetic effect, it is characterised in that：
Described medicine, cosmetics or the Periplapeptide A containing effective dose or America in daily chemical product
The total polypeptide extracts of big Lian, balance of auxiliary material or other compatible medicines.
8. promotion organization according to claim 7 is repaired novel polypeptide or the total polypeptide extract of American cockroach
Application in the product for preparing with tissue repair or improving skin aesthetic effect, it is characterised in that：
Described auxiliary material is solvent, disintegrant, flavouring, preservative, colouring agent or adhesive.
9. promotion organization according to claim 6 is repaired novel polypeptide or the total polypeptide extract of American cockroach
Application in the product for preparing with tissue repair or improving skin aesthetic effect, it is characterised in that：
The form of described medicine, cosmetics or daily chemical product is tablet, capsule, injection, solid fat
Matter nano particle, controlled release agent, gel ointment, ointment, externally-applied liniment, patch, creme, detergent,
Emulsion, gel or toner.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108158938A (en) * | 2018-02-13 | 2018-06-15 | 云南诺漫斯生物科技有限公司 | A kind of Pimple removing scream of the ingredient containing American cockroach and preparation method thereof |
| CN108703933A (en) * | 2018-05-12 | 2018-10-26 | 大理大学 | A kind of antibacterial skin moistening liquid soap and preparation method thereof |
| CN109966319A (en) * | 2017-12-22 | 2019-07-05 | 四川好医生攀西药业有限责任公司 | A kind of raw material of Kangfuxin Liquid and its preparation method and application |
| CN110609110A (en) * | 2019-09-24 | 2019-12-24 | 暨南大学 | Method for measuring content of tissue repair factor PA1 in periplaneta americana and related products thereof |
| CN110672737A (en) * | 2019-09-29 | 2020-01-10 | 浙江京新药业股份有限公司 | Method for identifying American cockroaches |
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| CN111484549A (en) * | 2019-01-28 | 2020-08-04 | 暨南大学 | Periplaneta americana tissue repair factor PA1 and application thereof |
| CN112457377A (en) * | 2020-12-07 | 2021-03-09 | 梅州市华师昆虫发育生物学与应用技术重点实验室广梅园研发中心 | Periplaneta americana polypeptide and application thereof |
| CN114152584A (en) * | 2020-09-08 | 2022-03-08 | 四川好医生攀西药业有限责任公司 | Method for detecting quality of periplaneta americana ethanol extract based on biological activity detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102743741A (en) * | 2012-05-06 | 2012-10-24 | 大理学院 | Preparation of blattodea polypeptide, and uses of the same in anti-gram-positive bacteria and anti-gram-negative bacteria |
-
2015
- 2015-11-11 CN CN201510768410.5A patent/CN106699849B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102743741A (en) * | 2012-05-06 | 2012-10-24 | 大理学院 | Preparation of blattodea polypeptide, and uses of the same in anti-gram-positive bacteria and anti-gram-negative bacteria |
Non-Patent Citations (1)
| Title |
|---|
| 蒋文贤: "美洲大蠊的化学成分", 《暨南大学学报》 * |
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