CN101157804A - Method for extracting melanin from black-bone chicken - Google Patents
Method for extracting melanin from black-bone chicken Download PDFInfo
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- CN101157804A CN101157804A CNA2006101698016A CN200610169801A CN101157804A CN 101157804 A CN101157804 A CN 101157804A CN A2006101698016 A CNA2006101698016 A CN A2006101698016A CN 200610169801 A CN200610169801 A CN 200610169801A CN 101157804 A CN101157804 A CN 101157804A
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Abstract
The invention relates to a melanin extraction method from a black-bone silky fowl, which comprises the following steps: the black-bone fowl is cut up and washed by using 1 M to 10 M of hydrochloric acid solution for 5 minutes to 30 minutes and is immerged into liquid nitrogen for 0.5 minute to 5 minutes; after that, the black-bone fowl is grinded into 60 meshes to 100 meshes powder and transferred into a Soxhlet extractor to do thermal reflux for 1.5 hours to 3 hours by using 1 M to 10 M of hydrochloric acid; after the leaching liquor is cooled to 0 DEG C to 5 DEG C to be precipitated; acetone distilled water, which has the same volume with the leaching liquor, is respectively added into the precipitate for washing for 3 to 4 times, drying, then the melanin is made. Through the grinding process after the liquid nitrogen is frozen, the method of the invention which can fully and effectively damage the structure of melanophore in tissue under the precondition of no changing the physical chemistry properties of the melanin, is in favor of fast melanin release, shortens melanin extraction time and keeps nearly complete the melanin morphological structure of the black-bone fowl.
Description
Technical field
The present invention relates to melanic extracting method, specifically, relate to a kind of melanic method of from Gallus Domesticus, extracting.
Background technology
Gallus Domesticus is the distinctive rare bird of China, has special nourishing and pharmaceutical use.In Compendium of Material Medica, LI Shi-Zhen is said " Gallus Domesticus has Bai Maowu bone person, black wool crow bone person, marble crow bone person, all black person of kindred, the white black bone person of meat is arranged, but see the black person of chicken tongue, and then kindred is all black, and it is better to be used as medicine ".As seen ancient medicine is when distinguishing the medicinal effect of Gallus Domesticus, is how much to be major criterion with the melanochrome in its meat, the bone.Modern medicine thinks that also the pharmaceutical use of Gallus Domesticus is its intravital melanochrome material.Melanochrome is the erratic polymer of a kind of complexity, extensively is present in many organisms and the tissue.The melanochrome in animality source is synthetic in melanosome, and melanosome is to be produced by the melanocyte that is distributed in skin or other tissue.Melanocyte produces two kinds of distinct melanochrome types, and a kind of is the eumelanin of brownish black, and a kind of is phaeomelanin russet.Experimental results demonstrate that both at home and abroad melanochrome can effectively protect biological organism by being permitted different ways, for example melanochrome is to ultraviolet absorption, to huge legendary turtle cooperation usefulness, removing free radical and anti-oxidant function of heavy metal ion or the like.Because its special property, melanochrome has been widely used in medical science, pharmacology, makeup and other field.
Difference according to melanochrome source obtains melanic method and also is not quite similar, and for example uses the method for chemosynthesis, method that acid solution is extracted, method that alkali lye extracts and microorganism synthetic method or the like.Yet the standard method of the none extraction purifying melanin of generally acknowledging up to the present.Along with to the going deep into of aspect researchs such as melanic medical science of Gallus Domesticus and pharmacology, press for set up fast, the extracting method of cheap, fidelity, from Gallus Domesticus, extract melanochrome expeditiously.
Up to the present, melanic method mainly contains two kinds in the extraction Gallus Domesticus: 1) Liu Wangyi used the method for hydrochloric acid degrade proteins to separate Gallus Domesticus melanochrome in 1979.Main process is: the sample with Gallus Domesticus is cut into fragment earlier, soaks with concentrated hydrochloric acid, repeats repeatedly, and the gained filter residue heats with the 6M hydrochloric acid reflux, and the time is about 300 hours, and collecting precipitation is with distilled water and washing with acetone drying.Shortcoming is that the time of hot hydrochloric acid reflux is oversize, destroys easily and changes melanic chemical structure and microscopic pattern; 2) the zymyhydrolyzed protein matter used in 2005 of people such as Cai Huazhen is separated Gallus Domesticus melanochrome.Mainly process is: earlier the Gallus Domesticus sample is rubbed, use aspartic protease then as lytic enzyme, and enzyme concentration 3.8%, 34.8 ℃ of control pH=2.8, hydrolysis temperatures, the time is about 20 hours, collecting precipitation, with ether, distilled water repetitive scrubbing, the dry melanochrome that gets.Advantage is can not destroy melanic form and structure substantially, and it is little to extract melanic loss.Shortcoming is the cost height, the enzymatic hydrolysis condition complexity, and complex steps, it is long to extract the melanic time.
Summary of the invention
The objective of the invention is to overcome in the existing Gallus Domesticus and extract the defective of melanochrome technology, thus provide a kind of step simple, fast, high-fidelity, melanochrome extracting method cheaply.
In order to realize purpose of the present invention, the invention provides a kind of melanic method of from Gallus Domesticus, extracting, comprise Gallus Domesticus chopping back is cleaned with hydrochloric acid soln, immerse liquid nitrogen and extract after 0.5～5 minute.
Gallus Domesticus chopping back was preferably cleaned 5～30 minutes with the hydrochloric acid soln of 1～10M.
Extraction behind the immersion liquid nitrogen is preferably the Gallus Domesticus behind the immersion liquid nitrogen is ground into 60～100 order powder, change in the apparatus,Soxhlet's, with the hydrochloric acid soln thermal backflow of 1～10M 1.5～3 hours, can stop, vat liquor is cooled to 0～5 ℃ and separates out precipitation, elder generation adds and the isopyknic acetone degreasing washing of vat liquor 3～4 times in the precipitation, and adding and the isopyknic distilled water wash of vat liquor are 3～4 times then, are drying to obtain.
Wherein when carrying out the Soxhlet extraction, used hydrochloric acid soln amount does not have strict regulation, can satisfy reflux conditions and both can.The acetone that adds is identical with the hydrochloric acid soln amount of adding with the amount of distilled water.
Of the present inventionly from Gallus Domesticus, extract melanic method compared with prior art, its advantage is: by the method for pulverizing behind the liquid nitrogen freezing, under the prerequisite that does not change the melanochrome physicochemical property, can be fully the effective structure of melanocyte in the disorganize, help melanic snap-out release, shortened and extracted the used time of melanochrome.Specifically, hydrochloric acid degrade proteins method than Liu Wangyi, the time of hot hydrochloric acid reflux was reduced to 1.5-3 hour from original 300 hours, had also reduced the destruction of hydrochloric acid to melanin structure, had kept the morphological structure of Gallus Domesticus melanochrome near-complete.Than protease method, advantage be reduced cost, shortened extract the required time, the operation fairly simple.
Description of drawings
Fig. 1 liquid nitrogen is pulverized, the design sketch that melanin granule discharges is observed in the comparison of homogenate tissue freezing section.
The melanic EPR wave spectrum of Gallus Domesticus behind Fig. 2 purifying.
Gallus Domesticus melanochrome SEM/TEM image behind Fig. 3 purifying.
Among Fig. 1, A is that liquid nitrogen is pulverized the back frozen section; B is a frozen section behind the tissue homogenate.
Among Fig. 3, A is the melanic SEM image of Gallus Domesticus behind the purifying on the 100um yardstick; B is the melanic SEM image of Gallus Domesticus behind the purifying on the 1um yardstick, and the granular substance among the figure is exactly a melanin granule; C is the melanic TEM image of the Gallus Domesticus behind the purifying of observing on the 200nm yardstick, and the fine particles among the figure is an amino acid.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1:
From Gallus Domesticus, extract melanic method, comprise Gallus Domesticus chopping back was cleaned 10 minutes with the 6M hydrochloric acid soln, immersed liquid nitrogen 1 minute.Gallus Domesticus behind the immersion liquid nitrogen is ground into 80 order powder, change in the apparatus,Soxhlet's, hydrochloric acid soln thermal backflow with 6M can stop in 2 hours, used hydrochloric acid soln amount satisfies reflux conditions, vat liquor is cooled to 4 ℃ and separates out precipitation, elder generation adds and the isopyknic acetone degreasing washing of vat liquor 4 times in the precipitation, and adding and the isopyknic distilled water wash of vat liquor are 4 times then, are drying to obtain.
Embodiment 2
From Gallus Domesticus, extract melanic method, comprise Gallus Domesticus chopping back was cleaned 30 minutes with the 1M hydrochloric acid soln, immersed liquid nitrogen 5 minutes.Gallus Domesticus behind the immersion liquid nitrogen is ground into 100 order powder, change in the apparatus,Soxhlet's, hydrochloric acid soln thermal backflow with 1M can stop in 3 hours, used hydrochloric acid soln amount satisfies reflux conditions, vat liquor is cooled to 5 ℃ and separates out precipitation, elder generation adds and the isopyknic acetone degreasing washing of vat liquor 3 times in the precipitation, and adding and the isopyknic distilled water wash of vat liquor are 3 times then, are drying to obtain.
Embodiment 3
From Gallus Domesticus, extract melanic method, comprise Gallus Domesticus chopping back was cleaned 5 minutes with the 10M hydrochloric acid soln, immersed liquid nitrogen 0.5 minute.Gallus Domesticus behind the immersion liquid nitrogen is ground into 60 order powder, change in the apparatus,Soxhlet's, hydrochloric acid soln thermal backflow with 10M can stop in 1.5 hours, used hydrochloric acid soln amount satisfies reflux conditions, vat liquor is cooled to 0 ℃ and separates out precipitation, elder generation adds and the isopyknic acetone degreasing washing of vat liquor 3 times in the precipitation, and adding and the isopyknic distilled water wash of vat liquor are 3 times then, are drying to obtain.
Embodiment 4
The tissue samples of Gallus Domesticus is divided into two parts, and portion carries out homogenate with refiner to tissue; The method that another part tissue samples adopts liquid nitrogen to pulverize.Then two increments are originally carried out tissue freezing section's (HE dyeing) comparison respectively and observe the effect that melanin granule discharges.
Found that to handle than tissue homogenate and can better promote breaking and the enrichment (Fig. 1) of melanin granule of melanocyte by the liquid nitrogen material previously treated, and the material of handling forms sizeable broken end (60～100 order), is easier to soaking into of hydrochloric acid than the material after the homogenate.
Embodiment 5
Whether the residual Gallus Domesticus tissue samples after Soxhlet extract finished is with long-time once more (48 hours) lixiviate of hot hydrochloric acid, observe to also have the melanochrome continuation to organize the sample from Gallus Domesticus to discharge.
Found that the melanochrome that present method is extracted in the black-bone chicken is a kind of process of disposable snap-out release, though promptly extracting the back residue still is black and has melanic existence, but residue is being extracted again by similarity condition, even use the longer time, also not again lixiviate go out melanochrome.
Embodiment 6
The pure Gallus Domesticus melanochrome of gained is used the electron paramagnetic resonance spectrum (EPR) method, and (ElectronParamagnetic Resonance EPR) analyzes (Fig. 2), and used melanochrome sample heavily is 10.3mg.
The melanic epr signal of Gallus Domesticus that analytical results discovery this law is purified is within melanic scope, and be the asymmetric unimodal curve of first time derivative condition end, Δ H=5.813 Gauss, g=2.000047 does not have zero-field splitting, does not have inner hyperfine structure, illustrated that we put forward melanic verity, and do not have the existence at assorted peak in the spectrogram, and having reflected that also the melanochrome impurity that this law carries is less, purity is higher.
Embodiment 7
With the Gallus Domesticus melanochrome of gained with scanning electron microscope (Scanning ElectronMicroscopy, SEM) and transmission electron microscope (Transmission Electron Microscope TEM) carries out morphological analysis (Fig. 3).
Arnaud (1981) can modify and destroy the ultrastructure of melanosome by the process that electron microscope discovery long time high concentration acid solution is extracted, thereby changes melanic microscopic pattern.Using the melanochrome of present method gained can be found on the yardstick of 10um by the SEM photo, is blocks of solid, and corner angle are clearly demarcated.If again it is amplified to the yardstick of 1um, can find that in conjunction with the TEM photo these blocks of solid are closely to be assembled by the shaggy spherical particle of diameter between 100-200nm to form again.Be identical YANLIU (2002) and be described in melanic microscopic appearance in the cuttlefish, and its also with the human hair in melanin granule likeness in form, be the aggregate of spherical particle.These have all illustrated the melanic pervasive microscopic pattern of organism, have also proved the high-fidelity characteristics of present method.
Embodiment 8
Susumu once reported the melanic deposition distribution at Gallus Domesticus different tissues position in 1999: periosteum＞sexual gland=tracheae 〉=heart, liver, stomach, caecum, muscle, skin, the report of this and Liu Wei (2004) coincide.We have chosen the skin histology of different sites of Gallus Domesticus respectively as the sample of experiment, and the method for using the moisture ware to survey dry-matter is calculated the melanochrome extraction yield (table 1) that uses present method gained.
The melanochrome extraction yield that found that the skin histology of different sites differs bigger, but melanic extraction yield is about 0.89 ‰ (the product addition gained of the extraction yield at each position and weighting coefficient) in total skin, use the extraction yield 0.94 ‰ of enzyme process in skin suitable with Susumu, but reduced cost, shortened extract the required time, operation is fairly simple.
The melanochrome extraction yield of the skin histology sample of table 1, different sites
| Experiment material (skin histology) | Weight in wet base (g) | Dry weight (g) | Melanochrome heavy (mg) | Extraction yield (‰) |
| Trunk | 32.42 | 11.63 | 9.6 | 0.82 |
| Cockscomb | 6.07 | 1.84 | 1.7 | 0.92 |
| Shin and pawl | 18.88 | 5.84 | 15.2 | 2.60 |
Claims (3)
1. one kind is extracted melanic method from Gallus Domesticus, it is characterized in that Gallus Domesticus chopping back is cleaned with hydrochloric acid soln, immerses liquid nitrogen and extracts after 0.5～5 minute.
2. the method for claim 1 is characterized in that the back hydrochloric acid soln with 1～10M of Gallus Domesticus chopping cleaned 5～30 minutes.
3. method as claimed in claim 1 or 2, the Gallus Domesticus that to immerse behind the liquid nitrogen that is extracted as that it is characterized in that immersing behind the liquid nitrogen is ground into 60～100 order powder, change in the apparatus,Soxhlet's, with the hydrochloric acid soln thermal backflow of 1～10M 1.5～3 hours, vat liquor is cooled to 0～5 ℃ and separates out precipitation, adding and the isopyknic acetone distilled water wash of vat liquor are 3～4 times respectively, are drying to obtain.
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| CNB2006101698016A CN100528969C (en) | 2006-12-28 | 2006-12-28 | Method for extracting melanin from black-bone chicken |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106009767A (en) * | 2016-06-11 | 2016-10-12 | 江西倍得力生物工程有限公司 | Method for extracting melanin from Taihe black-bone silky fowl |
| CN109054435A (en) * | 2018-09-10 | 2018-12-21 | 中国农业大学 | A method of extracting melanin from black-bone chicken periosteum |
| CN113564217A (en) * | 2021-08-03 | 2021-10-29 | 深圳太太药业有限公司 | Defatted and decolored pure black-bone chicken powder and production method and application thereof |
-
2006
- 2006-12-28 CN CNB2006101698016A patent/CN100528969C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106009767A (en) * | 2016-06-11 | 2016-10-12 | 江西倍得力生物工程有限公司 | Method for extracting melanin from Taihe black-bone silky fowl |
| CN109054435A (en) * | 2018-09-10 | 2018-12-21 | 中国农业大学 | A method of extracting melanin from black-bone chicken periosteum |
| CN113564217A (en) * | 2021-08-03 | 2021-10-29 | 深圳太太药业有限公司 | Defatted and decolored pure black-bone chicken powder and production method and application thereof |
| CN113564217B (en) * | 2021-08-03 | 2024-04-09 | 深圳太太药业有限公司 | Defatted and decolored pure black-bone chicken powder and production method and application thereof |
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| CN100528969C (en) | 2009-08-19 |
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