CN103316328B - EGF (Epidermal Growth Factor) collaborative symbiont capable of maintaining activity of EGF for long term and preparation method of EGF collaborative symbiont - Google Patents
EGF (Epidermal Growth Factor) collaborative symbiont capable of maintaining activity of EGF for long term and preparation method of EGF collaborative symbiont Download PDFInfo
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Abstract
The invention relates to an EGF (Epidermal Growth Factor) collaborative symbiont capable of maintaining the activity of an EGF for a long term and a preparation method of the EGF collaborative symbiont, particularly relates to an EGF collaborative symbiont capable of maintaining the activity of the EGF for more than two years and a preparation method of the EGF collaborative symbiont and belongs to extraction fusing and activity maintaining technologies for biological special useful components. The preparation method of the EGF collaborative symbiont is essentially characterized in that that wild food and medicine plants, namely gymnostemma pentaphylla, which are distributed in Xishuangbanna, Yunnan, are adopted, an extract of gymnostemma pentaphylla is fused with the EGF so as to form the EGF collaborative symbiont (EGF-c), and then, the activity retention time of the EGF is prolonged remarkably. Compared with the activity retention time of the current commercially available EGF raw materials, the activity retention time is prolonged to more than two years from seven days, so that the international difficult problem that the activity retention time of the EGF is short is solved, and a broad prospect is opened for the promotion, development and application of the EGF which is a significant result winning the Nobel biology-medicine prize.
Description
Technical field
The present invention relates to a kind of collaborative homobium of EGF that can keep for a long time epithelical cell growth factor EGF activity and preparation method thereof, particularly keep EGF activity to a two year above EGF to work in coordination with homobium and preparation method thereof, belong to extraction fusion and the activity keeping technology of biological special useful component.
Background technology
From Stanley Cohen Bo scholar in 1986 because finding EGF (Epidermal growth factor, hereinafter to be referred as EGF) and since obtaining Nobel's biology and Medicine, EGF research boom for a long time and has widely been risen in the whole world, research shows: EGF energy intense stimulus Growth of Cells, suppressing aging gene expresses, stop skin aging, make skin respectively form part best physiological status of maintenance.In addition, its can also irritation cell outer macromolecular synthetic and secretion, skin care, is to determine skin vitality and healthy key factor.And the reparation of burn and scald, skin injury is had to significant curative effect.Result of study further confirms EGF each system to human body, comprising: skeletal system, digestive system, blood system, respiratory system, hormonal system, reproductive system, immune system, nervous system etc. have good effect.
For a long time, owing to extracting the shortage of resources of EGF, obtain through refining difficulty, high cost, is difficult to extensive use aborning.By research, available technique for gene engineering gets, but cost is still higher.But more seriously the activity keeping of EGF is very difficult, no matter extract the EGF obtaining from human secretion, animal, plant or genetic engineering, all only have the activity keeping phase about seven days, this is that EGF promotes the biggest problem in production application.Study for a long period of time to so far, in production application, people only have the method for employing lyophilized powder that EGF is deposited separately to keep active, when use, will after EGF lyophilized powder and other substrate dissolution with solvents, within seven days, be finished temporarily.
Activity keeping difficulty, becomes the serious hindrance of the great achievement of this acquisition Nobel Prize in Application and Development.
Summary of the invention
The object of the invention is to overcome above-mentioned prior art deficiency, invention is a kind of from endemic plant soma, extraction effective ingredient and EGF synergism, form a stable collaborative body (EGF-c) of EGF, makes the active holding time of EGF from the original preparation method extending to more than 2 years for seven days.The product that the EGF-c making produces, has effect clear and definite, stable, can keep for a long time the advantages such as activity, for the Application and Development of EGF is opened up Liao Xin road.
The present invention also aims to adopt the ecological principle of distinctive collaborative symbiosis between biology, adopt plant material and EGF to form collaborative homobium, the active time of EGF was extended to more than 2 years from seven days, made EGF biological raw material EGF-c, can replace now commercially available epithelical cell growth factor product (as EGF lyophilized powder), for the manufacture raw material of medicine, cosmetics, health product, functional food etc., and have long, curative effect of active time clearly, the advantage such as effect stability.
Technical solution of the present invention is: a kind of collaborative homobium of EGF that can keep for a long time epithelical cell growth factor EGF activity, it is characterized in that: adopt wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli, its extract and EGF are merged, and forming the collaborative homobium of an EGF is EGF-c.
Described wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli is that the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli that is distributed in In Xishuangbanna of Yunnan is Gymnostemma pentaphylla.
A kind of collaborative homobium preparation method of EGF that can keep for a long time epithelical cell growth factor EGF activity, it is characterized in that: the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli that employing is distributed in In Xishuangbanna of Yunnan is Gymnostemma pentaphylla, the extract of its mao of leaf Herb Gynostemmae Pentaphylli and EGF are merged, and forming the collaborative homobium of an EGF is EGF-c.
The extracting method of the extract of the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli of described In Xishuangbanna of Yunnan is as follows:
By plant material hair leaf Herb Gynostemmae Pentaphylli, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10, soaks more than 300 days, by extracting liquid filtering under low temperature environment, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, with 5 times of petroleum ether to level of residue or chloroform washing and filtering, in the water-bath of 80 DEG C～90 DEG C, steaming desolventizes and is extracted thing.
The preparation method that can keep for a long time the collaborative body biological raw material (EGF-c) of epithelical cell growth factor of the collaborative homobium of EGF of epithelical cell growth factor EGF activity, is characterized in that containing following processing step:
1) extract extract from the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli of In Xishuangbanna of Yunnan:
By plant material hair leaf Herb Gynostemmae Pentaphylli, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10, soaks more than 300 days, by extracting liquid filtering under low temperature environment, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, with 5 times of petroleum ether to level of residue or chloroform washing and filtering, in the water-bath of 80 DEG C～90 DEG C, steaming desolventizes and is extracted thing;
2) by 1) extract the extract that obtains and mix with EGF, shake 60min in 37 DEG C of constant-temperature tables.After mix homogeneously at 15 DEG C of 8000rpm through centrifugal more than 1 hour, add the sodium lauryl sulfate of gross weight 0.01% and 0.015% polyoxyethylene sorbitan monooleate dehydration, after use homogenizer, homogeneous 15 minutes of high speed, in 3-5 degree constant temperature Celsius, leave standstill after 24 hours, obtain the collaborative body (EGF-c) of epithelical cell growth factor and pack stored for future use in handtailor container into.
The collaborative body biological raw material (EGF-c) of epithelical cell growth factor is applied to the functional living being raw material of medicine, cosmetics, health product or functional food.
In such use, keep the biological synergetic body EGF-c of EGF activity in various productions, to use content to be:
During medicine is made and used, EGF-c proportion is the 0.3-0.5% of product gross weight;
During cosmetics use, EGF-c proportion is the 0.10-0.15% of product gross weight;
During health product use, EGF-c proportion is the 0.15-0.25% of product gross weight;
During functional food uses, EGF-c proportion is the 0.10-0.20% of product gross weight.
The present invention keeps the biological synergetic technology of EGF activity, its feature adopts the wild food and medicament dual-purpose plant < hair leaf Herb Gynostemmae Pentaphylli >(Gymnostemma pentaphylla that is distributed in In Xishuangbanna of Yunnan in essence), its extract and EGF are merged, form the collaborative homobium (EGF-c) of an EGF, make the activity keeping time significant prolongation of EGF.Than the activity keeping time of existing commercially available EGF raw material, extended to more than 2 years from seven days.
The present invention is according to the ecological principle of ubiquitous collaborative symbiosis between biology, invent with epithelical cell growth factor (Epidermal growth factor, EGF) collaborative element and epithelical cell growth factor merge, form stable biological synergetic body, i.e. " epithelical cell growth factor is worked in coordination with body (EGF-c) " (hereinafter to be referred as EGF-c), make the EGF activity keeping time within internationally recognized about seven days, extending to more than 2 years at present, solve short this international difficult problem of EGF activity keeping time, for the Application and Development of the great achievement of this acquisition of EGF Nobel biology-Medicine, bright prospects are opened up.
Brief description of the drawings
Fig. 1 is EGF-c standard curve of the present invention.
Detailed description of the invention
The present invention will be distributed in the hair leaf strand glue orchid of In Xishuangbanna of Yunnan, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10.Under low temperature state environment, soak more than 300 days, by extracting liquid filtering, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, extremely colourless for several times with 5 times of petroleum ether to level of residue or chloroform washing, in the water-bath of 80 DEG C～90 DEG C, steaming is preserved stand-by after desolventizing.
By stand-by preparation obtained above, by a certain percentage, after mixing with EGF, shake 60min in 37 DEG C of constant-temperature tables.After mix homogeneously through centrifugal (15 DEG C of temperature, rotating speed 8000rp, time are more than 1 hour), add the sodium lauryl sulfate of gross weight 0.01% and 0.015% polyoxyethylene sorbitan monooleate dehydration, after use homogenizer, homogeneous 15 minutes of high speed, in 3-5 degree constant temperature Celsius, leave standstill after 24 hours, pack stored for future use in handtailor container into.
The present invention can be directly used in the function raw material of making medicine, cosmetics, health product, functional food, has use safety, the advantages such as effect is clear and definite, the activity keeping time is long, stable, cheap, abundant raw material.The ratio that the present invention uses in essence in various productions is: during medicine is made and used, EGF-c proportion, is generally the 0.3-0.5% of product gross weight.During cosmetics use, be generally the 0.1-0.15% of product gross weight.During health product use, be generally the 0.15-0.25% of product gross weight, during functional food uses, be generally the 0.10-0.20% of product gross weight.
Embodiment:
In order further to set forth technological means of the present invention and effect, below in conjunction with preferred embodiment, the inventive method is described in detail as follows, but content of the present invention is not limited to following content.
The present invention keeps EGF activity to the two year above collaborative homobium of EGF, adopts wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli, and its extract and EGF are merged, and forming the collaborative homobium of an EGF is EGF-c.
Described wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli is that the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli that is distributed in In Xishuangbanna of Yunnan is Gymnostemma pentaphylla.
The wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli that the present invention's employing is distributed in In Xishuangbanna of Yunnan is Gymnostemma pentaphylla, and its extract and EGF are merged, and forming the collaborative homobium of an EGF is EGF-c.
The extracting method of the extract of the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli of described In Xishuangbanna of Yunnan is as follows:
The present invention is by 1000 grams of plant material hair leaf Herb Gynostemmae Pentaphyllis, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10, soaks more than 300 days, by extracting liquid filtering under low temperature environment, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, with 5 times of petroleum ether to level of residue or chloroform washing and filtering, in the water-bath of 80 DEG C～90 DEG C, steaming desolventizes and is extracted thing.
What keep EGF activity works in coordination with body biological raw material (EGF-c) preparation method to more than 2 years epithelical cell growth factors, contains following processing step:
1) extract extract from the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli of In Xishuangbanna of Yunnan:
By 1000 grams of plant material hair leaf Herb Gynostemmae Pentaphyllis, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10, soaks more than 300 days, by extracting liquid filtering under low temperature environment, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, with 5 times of petroleum ether to level of residue or chloroform washing and filtering, in the water-bath of 80 DEG C～90 DEG C, steaming desolventizes and is extracted thing;
2) by 1) extract the extract that obtains and mix with EGF, shake 60min in 37 DEG C of constant-temperature tables.After mix homogeneously at 15 DEG C of 8000rpm through centrifugal more than 1 hour, add the sodium lauryl sulfate of gross weight 0.01% and 0.015% polyoxyethylene sorbitan monooleate dehydration, after use homogenizer, homogeneous 15 minutes of high speed, in 3-5 degree constant temperature Celsius, leave standstill after 24 hours, obtain the collaborative body (EGF-c) of epithelical cell growth factor and pack stored for future use in handtailor container into.
The collaborative homobium of EGF is the functional living being raw material of EGF-c for medicine, cosmetics, health product and functional food.
The ratio that keeps the biological synergetic body EGF-c of EGF activity to use in various productions is: during medicine is made and used, EGF-c proportion, is the 0.3-0.5% of product gross weight;
Cosmetics are the 0.1-0.15% of product gross weight in using;
Health product are the 0.15-0.25% of product gross weight in using;
Functional food is the 0.10-0.20% of product gross weight in using.
The discriminating of above-mentioned experiment:
1. the standard curve of the collaborative body (EGF-c) of epithelical cell growth factor is formulated:
This product is the raw material preparation that biological species contains polypeptide, adopts the correlated characteristic of gel permeation chromatography (Sephadex) to detect data as the standard of sample, can effecting reaction raw material preparation in the distribution of the collaborative thing such as polypeptide and Yellow ketone.
Experiment flow:
(1) it is 15% for subsequent use material liquid being adjusted to concentration.
(2) by for subsequent use tris-HCl buffer (0.1mol/L, ph=8.0) balance for the sephadex column of G-75.
(3) by centrifugal 10min under the material liquid 12000r/min getting ready, abandon precipitation, get 2ml loading, with tris-HCl buffer (0.1mol/L, ph=8.0) eluting, flow speed control is at 0.3ml/min; Sample is collected, and every pipe meets sample amount 3ml.
(4) connect sample until reserve ultraviolet value that sample records under wavelength 280nm in sephadex column lower than 0.1, stop collecting.
(5) sample collection being obtained, by acquisition time label, is measured the ultraviolet value under wavelength 280nm every a pipe, and taking pipe number as transverse axis, ultraviolet value is that the longitudinal axis is figure.
Now by the digital actual measurement attached (see photo) of canonical plotting that arranges and cut off the collaborative body of epithelical cell growth factor of making
In the collaborative body biological raw material (EGF-c) of epithelical cell growth factor, be no matter benzoyl (220-280nm) in collaborative body and the absorption of cinnamyl (300-400nm), or protein polypeptide is in the absorption at 280nm place, is all peak value.Collection of illustrative plates has fully reflected the situation that is related to of working in coordination with body and two factors of epithelical cell growth factor (EGF) in (EGF-c).
As can be seen from Figure, the contained albumen of sample and after gel permeation chromatography shown ultraviolet characteristic spectrum kept stable, can detect index as of raw material EGF-c.
Peak-peak is stabilized between 1.9-2.5.(see photo)
Peak type constant is concluded, and each peak starts, peak value arrives, all keep better stability between end zone.
| I peak starts | I peak-to-peak value | I finishes at peak | II peak starts | II peak-to-peak value | II finishes at peak |
| 99—116 | 120—146 | 149—172 | 212—251 | 252—292 | 290—321 |
Material sample operates by this experiment flow, and measured ultraviolet data should meet above scope of data after being figure in accordance with the law, when result difference is little, can judge raw material conformance with standard (seeing accompanying drawing 1).
2. the quantitative assay of EGF in the collaborative body EGF-c of epithelical cell growth factor
Pre-treating method: trichloroacetic acid (TCA) acetone precipitation
Experiment flow:
(1) material liquid is concentrated, get supernatant after centrifugal for subsequent use;
(2) the material liquid sample of learning from else's experience centrifugal, adds the extracting solution (10% TCA) of 3 times of volumes, is placed in-20 DEG C and spends the night;
(3), in 12000rpm/min, centrifugal 10min, abandons supernatant, collecting precipitation;
(4) precipitation adds isopyknic ice bath acetone, mixes rear centrifugal (12000rpm, 10 minutes), abandons supernatant vacuum drying, saves backup;
(5) before loading, add phosphate buffered solution (pbs) sample dissolution of 1.6 times of volumes stand-by.
Testing process:
(1) after collection of specimens, extract as early as possible, extract and undertaken by pertinent literature, after extraction, should test as early as possible.If can not test at once, specimen can be put in to-20 DEG C of preservations, but should avoid multigelation.
(2) can not detect the sample containing NaN3, because of (HRP) activity of NaN3 inhibition horseradish peroxidase.
Operating procedure
(1) dilution of standard substance: one of former times of standard substance dilute by the following method in small test tube.
The former times of standard substance of No. 5 standard substance of 120ng/L 150 μ l add 150 μ l standard substance diluents
No. 5 standard substance of No. 4 standard substance 150 μ l of 60ng/L add 150 μ l standard substance diluents
No. 4 standard substance of No. 3 standard substance 150 μ l of 30ng/L add 150 μ l standard substance diluents
No. 3 standard substance of No. 2 standard substance 150 μ l of 15ng/L add 150 μ l standard substance diluents
No. 2 standard substance of No. 1 standard substance 150 μ l of 7.5ng/L add 150 μ l standard substance diluents
(2) application of sample: establish respectively blank well (blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), gauge orifice, testing sample hole.The accurate application of sample 50 μ l of standard substance on the coated plate of enzyme mark, first add sample diluting liquid 40 μ l, and then add testing sample 10 μ l (the final dilution factor of sample is 5 times) in testing sample hole.Sample is added on bottom, ELISA Plate hole by application of sample, do not touch hole wall as far as possible, rocks and mix gently.
(3) incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.
(4) dosing: by for subsequent use after 30 times of dilutions of 30 times of concentrated cleaning solutions use distilled water
(5) washing: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.
(6) enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
(7) incubation: operation is with 3.
(8) washing: operation is with 5.
(9) colour developing: every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 DEG C of lucifuges develop the color 15 minutes.
(10) stop: every hole adds stop buffer 50 μ l cessation reaction (the now blue vertical yellow that turns).
(11) measure: with blank air-conditioning zero, 450nm wavelength is sequentially measured the absorbance (OD value) in each hole.Mensuration should be carried out in 15 minutes adding after stop buffer.
(12), according to absorbance (OD value), can draw concrete the contain numerical quantity of EGF in collaborative body.
3. the qualification of collaborative element c in EGF-c
3.1 measure the selection of wavelength; Accurately take to be fully dried to rutin standard reagent 0.0050g 60 dissolve with ethanols of constant weight and to proceed to completely in 50mL volumetric flask and shake up to obtain the concentration rutin standard solution that is 0.lmg/mL with 60 ethanol standardize solution.Accurately draw above-mentioned rutin standard solution 1.0mL mends to add 0.3mL 5W/V sodium nitrite solution to shake up to place to 5.0mL with 60 ethanol and adds after 6min 0.3mL 10 W/V nitric acid companion solution to place after 6min add 4.0mL 1mol/l sodium hydroxide solution to mix to add the ethanol room temperature placement 15min of 0.4ml 60 with 60 ethanol dilutions to scale again to measure its absorbance taking 60 alcoholic solution as blank reference again in 10mL scale test tube.From figure, draw maximum absorption wavelength at 400nm to 600nm scope interscan wavelength.
The preparation of 3.2 standard solution is drawn respectively above-mentioned rutin standard solution 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL and be carried out at its light absorption value of wavelength 510nm place's colorimetric determination taking 60% alcoholic solution as blank reference by 1.1 step in 6 10mL scale test tube.With map to obtain standard curve and the equation of linear regression of rutin concentration C and absorbance A of rutin concentration C and absorbance A.
The mensuration of 3.3 sample general flavone contents get Different Extraction Method gained sample liquid 1.0mL in 10mL scale test tube by 2.1 in subsequent step be carried out at its light absorption value of wavelength 510nm place's colorimetric determination and calculate total flavones yield according to formula taking the liquor-saturated solution of 60 second again as then blank reference calculates total flavones amount in every milliliter of extracting solution by 2212 equations of linear regression that obtain.
Total flavones yield %=(CV
1v
210
-3)/(WV
0) 100
In formula: C: the concentration mg/mL that measures sample liquid;
V0: the volume mL that measures absorbance sample liquid used;
V1: dilute volume mL when mensuration;
V2: volume mL after sample liquid standardize solution;
W: sample quality g.
4. the pharmacodynamics of collaborative element
Be present in the collaborative body of EGF-C(epithelical cell growth factor with cooperative mode) in flavone compound, through studies confirm that in a large number, there is medicinal effects widely, for example: to protecting the effects such as cardiac muscle, anticoagulation, arrhythmia obviously.Digestive system is had to raising Small Intestinal, significantly suppress external ileum contraction motion.Stress ulcer is had to protective effect, alleviate mucosal lesion, suppress the formation of gastric ulcer.Antiinflammatory and anti-immunization aspect, Scutellaria baicalensis stem-leaf total flavonoid can reduce rat paw edema due to fresh albumen.Herba Senecionis Scandentis total flavones xylol causes mice auricle swelling model, glacial acetic acid is caused mouse peritoneal capillary permeability model, all has obvious antagonism to mice granuloma induced by implantation of cotton pellets model with to air sac models of synovitis in mice.Radix Astragali total flavones has therapeutical effect to arthritis.Folium Ginkgo total flavones 100mg/kg can obviously suppress the scorching mice ear edema of caused by dimethylbenzene xylene, and 50,100mg/kg can suppress Ovum Gallus domesticus album and cause the swelling of rat foot claw. Fructus Pruni pseudocerasi total flavones has obvious inhibitory action to rabbit ear cold injury inflammation.Antitumor action aspect, flavone compound has good anti-tumor activity, and icariin is the potent inhibitor of tumor cell telomerase activation.In Radix Glycyrrhizae, Flavonoid substances has phytoestrogen activity, and luteolin has very strong liver cancer apoptosis reducing activity.Herba Moslae Scabrae total flavones energy Dichlorodiphenyl Acetate induced mice pain has remarkable analgesic activity.Folium Ginkgo total flavones can all have obvious analgesic activity to formaldehyde in mice method, hot plate method, warm bath method, the caused induced pain reaction of writhing method.In leaf of Rhizoma Belamcandae, mangiferin has good antipyretic effect.Carica papaya flavonoids Dichlorodiphenyl Acetate, temperature induced mice pain have good analgesic activity. and various flavone compounds all have protective effect in various degree to Experimental Hepatic Damages such as drug induced hepatic injury, alcoholic liver injury, chemical liver injury, j immunologic liver injury as flavonoid, flavonols, flavanone, osajin, flavanone etc.Flavone causes mouse brain memory represents obstacle to ethanol and improves significantly, and can significantly improve recent memory and the longterm memory function of mice.Herba Epimedii total flavones can delay nematicide aging, and can significantly improve its acute heat stress under stress ability. propolis flavone can reach the object of slow down aging.Total flavones is in antidepressant, and improving immunologic function all has very good effect.
Epithelical cell growth factor is worked in coordination with the toxicology test of body (EGF-c)
Epithelical cell growth factor is worked in coordination with body (EGF-c), be to the human body EGF factor without any toxicity intrinsic by human body itself, and the plant material synergism of food and medicament dual-purpose forms, should belong in theory nontoxic, but in order further to prove that it is nontoxic, spy has carried out this acute toxicity test.
One. summary:
This test adopts ICR mice to carry out the heavy dose of single administration acute toxicity test of its mouse oral to EGF-c.Cheat no abnormality seen after medicine.Its measurement result: MTD is 26.4g/kg body weight.
Two. test objective: observation once gives by EGF-c heavy dose the acute toxic reaction and the death condition that produce after animal.
Three. material:
1. tested material: EGF-c 5.0g/ bag, each 1 bag, every day three times.Lot number: 20000318.Reaching ecological product company by Kunming Lv Yuan provides.
2. laboratory animal: ICR mice, is provided by animal housing of natural drug pharmacology key lab of unming Medical College.The animal quality certification: No. 9804,9806, the real animal mat woven of fine bamboo strips in Yunnan.
Four. method:
Selecting body weight is 20 of the healthy ICR mices of 18-22g, and male and female half and half take EGF-c medicated powder 33g and add warm water and become sepia thick to 50ml, and by 0.4ml/10g body weight gastric infusion, Continuous Observation seven days after administration, remembers the poisoning and death condition of Pearl animal.
Five. result:
After 20 mouse stomach administrations, seven days there is not any untoward reaction in Continuous Observation animal, and all animals survival is all right, and freely, feed, drinking-water are normally in activity.It is 26.4g/ kg body weight that result records EGF-c its mouse oral MTD, calculates by 60 kg body weight, is 105.5 times of clinical recommended drug amount (0.25g/ kilogram).
Six: evaluate:
This research department carries out its mouse oral mtd test with EGF-c, and its MTD is 26.4g body weight.Prompting EGF-c its mouse oral.。LD50 is greater than 26.4g/ kg body weight, is calculated as 105.5 times of clinical recommended drug amount by 60 kg body weight.
The activity keeping test of the collaborative body EGF-c of epithelical cell growth factor
This test adopts respectively following two kinds of methods to carry out
One. mtt assay is measured the activity of different storage lives under EGF-c room temperature: concrete grammar is as follows:
1. cultivate human skin fibroblast (HSF), make its recovery.
2. by the HSF cell trypsinization of exponential phase, inoculating cell: with the culture fluid containing 10% tire calf serum the HSF cell suspension having digested, sucking-off trypsin after cell rounding.
3. after cell counting, seed cells into 96 orifice plates with 2000, every hole, every hole 200ul, establishes 3 parallel holes for every group.The hole of plate periphery does not add cell, has prevented edge effect.Blank hole zeroing, makes the active negative control of EGF-c not add the cell of EGF-c.
4. at 37 DEG C, 5%CO2
2incubator in cultivate after 24 h, add respectively EGF-c material sample and the contrast of various dose.Establish 3 parallel holes for every group, continue to cultivate 24h, add MTT solution 20ul to continue to cultivate 4 h,
5. stop cultivating, abandon culture fluid, add dimethyl sulfoxide (DMSO) 150ul/ hole, vibration, measures the light absorption value in each hole, and averages at enzyme-linked immunosorbent assay instrument OD490nm place, the results are shown in following table.
Discuss: in the situation that EGF reference substance just lost whole activity after seven days, it is stable that the activity of EGF-c keeps always, and the activity after 3 years still remains on more than 92%.
Note; 1. reference substance is commercially available EGF lyophilized powder, and its fresh sample activity value is 1.512, a little less than EGF-c sample.
2. blank activity is 0.514
2. the activity test that EGF-c recovers mice wound
Method: first test mice is anaesthetized with pentobarbital sodium solution, cut off respectively a size identical osculum of trying one's best in the both sides, back of each mice, Yong Xin Fresh EGF-c water preparation respectively, and preserve at normal temperatures the EGF-c water preparation after a year, be applied on wound, be used as blank with distilled water and be coated with, administration every day secondary, observes once for every two days.
Result:
Discuss: 1. EGF-c has positive effect to mice wound healing
2. the EGF-c placing after a year does not obviously reduce the effect of mice healing
The Pharmacodynamics of the collaborative body EGF-c biological raw material of EGF
Effect of EGF, is unanimously recognized that skin Chong wound, burn and scald, wound repair and timidly has remarkable result except cicatrix etc. by the world, below emphatically by EGF-c test observation effect in these areas in addition emphasis sum up:
1. skin trauma
Fresh wound, after use, 3-5 minute pain is eliminated, minor cut or wound 24-48 hour all healed, all healed in large wound 3-5 days, especially big wound 10-15 days all healed.The most magical is after wound healing scar not.Model case: 1. the worker Liu Zhengwei of Kunming post and telecommunications office, the palm of the hand is broken into muddy flesh by propeller, with EGF-c mono-month entirely more after, be hard to tell that hands injured, the lines on palm all recover as hinder front.2. the former director Zhao Yuening of Swam portion of botanical garden, Chinese Academy of Sciences Xishuangbanna trip, traffic accident face is injured, wipes a bulk of skin, does not entirely stay any scar after more with EGF-c.
2. burn, scald
The injured EGF-c that used in time at that time, no matter order of severity size, all pain relievings immediately, do not get blister and other any symptom such as red, swollen, hot.If while failing timely medication generation above-mentioned symptom, use after EGF-c, above-mentioned symptom rapidly disappears, and wound heals very soon, after same magical Shi Quan More, does not leave any cicatrix.Model case: the 1. electrician of Xishuangbanna Tropical Plant Garden, Chinese Academy of Sciences Zhang Ming, after being injured by high-tension electricity, an arm inflammation is suppurated, very fast complete after use EGF-c, does not leave any scar.2. the Xishan District, Kunming City light Xu Mei of restaurant English, pressure cooker accident, whole face is scalded red and swollen foaming, very fast complete after use EGF-c, does not only leave scar, and facial original speckle has all been removed.I say: " really because Woe obtains good fortune, done a second advanced cosmetic surgery.”
3. repair cicatrix
Due to the various cicatrixes that a variety of causes stays in history, adhere to using after EGF-c a period of time, can make cicatrix level and smooth and even disappearance gradually.Model case: the 1. workman of Kunming Transformer Factory Zhang Weichun, the serious baked wound of 30 years front faces, leaves very ugly shameful scar, used EGF-c after two months face greatly take on a new look, See people just says: " first half of one's life, I was ghost, and the latter half of one's life, I just really became people ".2. PLA Shijiazhuang information portion cadre's sanitarium China of Feng state, back car Woe leaves the large purple scar of palm, within 8 years, does not disappear, and with two weeks after EGF-c, cicatrix all disappears.
4. Shang Kou More closes
All kinds of wounds that can not heal for a long time, with healing very soon after EGF-c.Model case: former deputy secretary comrade Liang Jia of Yunnan Provincial Party committee, 89 years old now, the sick bed of being in hospital of Long phase, three centimeters of left and right wounds so that vertebra afterbody splits, medication is multiple can not close by More, can not sit, and can not put down sleeping, very painful.With having grown new meat after EGF-c, heal fine, can sit and within several hours, see TV.
Prove according to series of experimental results such as above-mentioned relevant pharmacodynamics, toxicology, activity, EGF-c is a novel EGF product, can replace now commercially available EGF commodity completely, as the making raw material of medicine, health product, cosmetics and functional food, and on activity keeping, considerably beyond existing EGF commercially available prod, thoroughly solve a great problem of this great achievement of EGF in applying.
The collaborative body biological raw material (EGF-c) of epithelical cell growth factor of the present invention is identical with the EGF selling in the market, have and promote metabolism, the reparation of quickening human body cell etc. of human epidermal cell by the peculiar wide application effect to human body of internationally recognized EGF, can be directly used in a kind of functional living being raw material of medicine, cosmetics, health product, functional food etc., these goods are safe and reliable, in order to replace now commercially available EGF product, and have and keep the active time more than 2 years, aboundresources, with low cost, effect really, the advantage such as stable.
Claims (5)
1. the collaborative homobium of EGF that can keep for a long time epithelical cell growth factor EGF activity, is characterized in that: adopt wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli, its extract and EGF are merged, forming the collaborative homobium of an EGF is EGF-c;
Wherein extracting extract step from wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli is:
1) by plant material hair leaf Herb Gynostemmae Pentaphylli, be immersed in special ceramic Sheng product with 75% edible ethanol, immersion ratio is 1:10, soaks more than 300 days, by extracting liquid filtering under low temperature environment, filtrate decompression distillation, reclaim ethanol, after residue is dry, add 5 times to the water dissolution of level of residue, with 5 times of petroleum ether to level of residue or chloroform washing and filtering, in the water-bath of 80 DEG C～90 DEG C, steaming desolventizes and is extracted thing;
By above-mentioned steps 1) extract the extract that obtains and mix with EGF, shake 60min in 37 DEG C of constant-temperature tables; After mix homogeneously at 15 DEG C, 8000rpm through centrifugal more than 1 hour, add the sodium lauryl sulfate of gross weight 0.01% and 0.015% polyoxyethylene sorbitan monooleate dehydration, after use homogenizer, homogeneous 15 minutes of high speed, in 3-5 DEG C of constant temperature, leave standstill after 24 hours, obtaining the collaborative body of epithelical cell growth factor is EGF-c, packs stored for future use in handtailor container into.
2. the collaborative homobium of the EGF that can keep for a long time epithelical cell growth factor EGF activity according to claim 1, is characterized in that: described wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli is that the wild food and medicament dual-purpose vegetable hair leaf Herb Gynostemmae Pentaphylli that is distributed in In Xishuangbanna of Yunnan is Gymnostemma pentaphylla.
3. the collaborative homobium of EGF that can keep for a long time epithelical cell growth factor EGF activity according to claim 1 and 2, is characterized in that: its functional living being raw material as medicine, cosmetics, health product or functional food uses.
4. the collaborative homobium of EGF that can keep for a long time epithelical cell growth factor EGF activity according to claim 1 and 2, is characterized in that: keep the biological synergetic body EGF-c of EGF activity in various productions, to use content ratio to be:
During medicine is made and used, EGF-c proportion is the 0.3-0.5% of product gross weight;
During cosmetics use, EGF-c proportion is the 0.10-0.15% of product gross weight;
During health product use, EGF-c proportion is the 0.15-0.25% of product gross weight;
During functional food uses, EGF-c proportion is the 0.10-0.20% of product gross weight.
5. the collaborative homobium of the EGF that can keep for a long time epithelical cell growth factor EGF activity described in claim 1 or 2 is in the application of preparing cosmetics, health product or functional food, and described epithelical cell growth factor uses as functional living being raw material.
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Non-Patent Citations (4)
| Title |
|---|
| 绞股蓝在系列化妆品中的应用;葛金庚;《科技与经济》;19950831(第4期);第1页 * |
| 绞股蓝的药理作用;肖东等;《中成药》;19920131;第14卷(第1期);第1-2页 * |
| 肖东等.绞股蓝的药理作用.《中成药》.1992,第14卷(第1期), |
| 葛金庚.绞股蓝在系列化妆品中的应用.《科技与经济》.1995,(第4期), |
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