CN1394875A - Method for extracting fimbrin - Google Patents

Abstract

The present invention discloses an extraction method of fimbrin which is a natural biological macromolecule with good oxygen osmolarity, good air permeability, excellent biological compatibility, good flexibility, good humoral moistening property and anti-pollution property and good stability. It is specially used for making contact lens. Said invented fimbrin pure solution also can be used for making ophthalmic bandage, and bandage and artificial skin for curing other diseases.

Description

A kind of extraction method of silk fibroin

Technical field:

The invention relates to a method for extracting natural macromolecular biopolymers, specifically, compared with all contact lens materials currently on the market, it belongs to a kind of biopolymer with good oxygen permeability, good air permeability, and excellent biological properties. It is a natural biopolymer with good body compatibility, good softness, good body fluid wettability and stain resistance, and good stability. This polymer is especially suitable for making soft contact lenses.

Background technique:

Studies have shown that for contact lenses, especially for contact lenses suitable for long-term wear, the materials that can be used for preparation must have the following characteristics:

(1), oxygen permeability;

(2), softness;

(3), hydrophilicity;

(4), stability;

(5), wettability of tears;

(6), anti-dehydration;

(7), water resistance;

So far, the development process of contact lens materials has been for a long time, mainly through the following development stages: the earliest application is the hard plastic material polymethyl methacrylate PMMA, although the contact lenses made of this material It has good stability and dehydration resistance, but is poor in oxygen permeability, softness, hydrophilicity, long-term tear wetness and anti-smudge performance; it is followed by hydrogel soft lenses, which are made of polymer Made of 2-hydroxyethyl methacrylate PHEMA and its related materials, this lens has been widely welcomed by people, but the fly in the ointment is that the oxygen permeability of the lens is too poor. Therefore, when wearing this kind of lens, the cornea will not receive sufficient oxygen supply, and some adverse reactions, such as corneal ulceration, inflammation, and thickening, will often occur. In response to this situation, people have developed the third generation of contact lens: HGP, which is generally made of acrylic polysiloxane and acrylic polyfluorosiloxane. Although this kind of lens greatly improves the oxygen transmission rate, it also has good stability and dehydration resistance, but it lacks softness, hydrophilicity, long-term tear wettability and anti-smudge resistance. Because of these defects, people who wear such contact lenses for a long time often feel uncomfortable eyes and find that the cornea is worn and deformed. At present, in order to prepare contact lenses that meet the above-mentioned various characteristics and are suitable for long-term wear, people are developing soft gas-permeable lenses SGP. For example: Zhang's US Patents 4,182,882 and 4,343,927; Tanaka's US Patent 4,139,513 and Leboeuf's US Patent 4,246,389. Although the lenses made according to these patents can have good oxygen permeability, softness and hydrophilicity, there are still defects in other properties.

Obviously, in order to prepare contact lenses that meet the above-mentioned required characteristics and are suitable for long-term wear, the technology of contact lenses should be raised to a new level, so that patients can really enjoy the benefits of such lenses without suffering harm. At present, we urgently need breakthroughs in materials, not just working on the modification of existing contact lens materials. After our research, we found that the development of silk fibroin materials may bring about a revolution in contact lens technology.

Scientific research shows that silk is composed of two parts: fibroin and sericin. Sericin is wrapped around silk fibroin, accounting for about 25% of the weight, and there are about 5% impurities in silk. , Silk fibroin is the main part of silk, accounting for about 70% of the weight. Silk fibroin is based on the antiparallel folded chain conformation (β-sheet), forming microfibrils with a diameter of about 10 nm, and countless microfibrils are closely combined to form thin fibers with a diameter of about 1 μm, and about 100 thin fibers are arranged along the long axis to form A single fiber with a diameter of about 10μm-18μm, that is, silk protein fiber. .

Silk fibroin contains 18 kinds of amino acids, and among them, alanine (Ala), glycine (Gly) and serine (Ser), which have relatively simple molecular formulas, contain relatively large amounts, accounting for more than 85% of the total amount. The molar ratio of the three It is 4:3:1. And according to a certain sequence structure (Ala-Gly-Ser) arrangement to form regular segments, most of these segments are located in the crystallization region of silk fibroin; and phenylalanine (Phe) with larger side groups, Tyrosine (Tyr), tryptophan (Try), etc. mainly exist in the amorphous region. The molecular weight of silk fibroin is very high, in the range of (3.6-3.7)×10 ⁵ .

The silk fibroin in the present invention is a kind of natural protein, which has porosity, high water absorption and moisture regain, good oxygen permeability, excellent biocompatibility, good body fluid wettability and stain resistance. Therefore, compared with synthetic high molecular polymers, contact lenses made of this material have excellent performance, and people's eyes will feel more comfortable when wearing it.

Invention content:

Silk fibroin in the present invention is a kind of natural biopolymer material that can be extracted from silk, and the extraction method of this silk fibroin solution, carries out according to the following steps:

1. Remove the silkworm chrysalis from the silkworm cocoons, wash and grind the silkworm cocoons for later use;

2. Sericin and impurities in silkworm cocoons can be removed by the following methods to obtain silk fibroin fibers:

(1) Organic acid method: Use organic acid-tartaric acid as a degumming agent, tartaric acid solubility is 12-18%, degumming at a temperature of 70-100°C.

(2) Soap cooking method: use a kind of Marseilles soap prepared from olive oil as a degumming agent, and degumming at 80-120°C.

(3) Alkali method: use one or both of Na ₂ CO ₃ and NaHCO ₃ as a degumming agent, the solubility of which is controlled at 0.2-6%, preferably 0.4-0.8%, at a temperature of 70-100°C Ungummed. Before degumming, it is better to soak the silkworm cocoons treated in step 1 at 20-60°C for 8-20 hours, and the degumming time of the soaked silkworm cocoons only needs 0.5-3h. Alkaline method is the best method.

3. Soak the silk fibroin fiber in deionized water, and then dry it at 30-100°C.

4. Dissolve silk fibroin fibers in the following solvents:

(1) Using 7-11M LiBr aqueous solution as a solvent to dissolve silk fibroin fibers at a temperature of 30-60°C. or

(2), using Cu-C ₂ H ₇ N, Cu(OH) ₂ -NH ₄ or [Cu(NH ₃ ) ₄ ](OH) ₂ aqueous solution as a solvent, at a temperature of 40-90 ° C, silk fibroin Fiber dissolves. or

(3) Use CaCl ₂ -H ₂ O or CaCl ₂ -C ₂ H ₆ OH ₂ O solution with a solubility of 35-60%, preferably 40-45% as a solvent, and dissolve at a temperature of 60-80°C. It is better to soak the silk fibroin fiber treated in step 3 at 20-50° C. for 8-20 hours before dissolving, and the dissolving time of the soaked silk fibroin fiber only needs 0.8-3 h. This solvent is optimal.

5. Dialyze the silk fibroin solution in a cellulose separation membrane to remove the influence of small molecules in the solvent, and then centrifuge to remove impurities, so as to obtain a pure silk fibroin solution.

The pure silk fibroin solution obtained in the present invention can be cured at 40-90°C, and the general curing time is 5-7 hours to obtain a transparent silk fibroin film with a certain thickness, which can be used as a new type of contact lens. Eye bandages for controlled drug release for eye diseases or protective devices for corneal injury or disease, bandages for wounds or artificial skin for burns.

Detailed ways:

Example 1

First, we remove the silkworm pupae from the cocoon, mince the cocoon and put it in a flask, use NaHCO ₃ solution as a degumming agent and soak it at 25°C for 8h, and its solubility is controlled at 0.6%. It is necessary to remove sericin at a temperature of 80°C 1.5h. Then the obtained silk fiber is washed with deionized water, and dried at a temperature of 60°C. Mince the silk fibroin fiber again and put it into a flask, add CaCl ₂ -H ₂ O solution with a solubility of 40% as a solvent and soak for 12 hours at 25°C, and dissolve the silk fibroin fiber at a temperature of 79°C, only 2.8 h, after cooling, put the silk fibroin solution in a cellulose separation membrane and dialyze for 3 days to remove the small molecules in the solvent, and then go through high-speed centrifugation to remove impurities, so as to obtain a pure silk fibroin solution. Solidify the silk fibroin solution at a temperature of 80°C to obtain the required silk fibroin glasses blank. After visible spectrum analysis, the light transmittance of this glasses blank is 94%; and the hydrophilic property is good, and the contact with water The angle is 23.5°.

Example 2

First, we remove the silkworm cocoons from the cocoons, mince the cocoons and put them in a flask, use Na ₂ CO ₃ solution as a degumming agent to soak for 12 hours at 25°C, and control its solubility to 0.4%, and remove the silk at a temperature of 72°C. Glue needs 1.8h. Then the obtained silk fiber was washed with deionized water, and dried at 68°C. Mince the silk fibroin fiber again and put it into a flask, add CaCl ₂ -H ₂ O solution with a solubility of 45% as a solvent, soak at 25°C for 18 hours, and dissolve the silk fibroin fiber at a temperature of 70°C, only need After about 2 hours, after cooling, put the silk fibroin solution in a cellulose separation membrane and dialyze for 4 days to remove small molecules in the solvent, and then remove impurities through high-speed centrifugation to obtain a pure silk fibroin solution. Solidify the silk fibroin solution at a temperature of 90°C to obtain the required silk fibroin glasses blank. After visible spectrum analysis, the light transmittance of this glasses blank is 98%; and the hydrophilic property is good, and the contact with water The angle is 22°.

Example 3

First, we remove the silkworm pupae from the cocoon, mince the cocoon and put it in a flask, use NaHCO ₃ solution as a degumming agent and soak it at 25°C for 8 hours, and its solubility is controlled at 1%. It is necessary to remove sericin at a temperature of 100°C 0.8h. Then the obtained silk fibers were washed with deionized water, and dried at a temperature of 76°C. Mince the silk fibroin fiber again and put it into a flask, add a CaCl ₂ -H ₂ OC ₂ H ₆ O solution with a solubility of 45% as a solvent and soak it at 25°C for 19h, and put the silk fibroin fiber at a temperature of 69°C It only takes 1.5 hours to dissolve. After cooling, put the silk fibroin solution in a cellulose separation membrane and dialyze for 4 days to remove small molecules in the solvent, and then go through high-speed centrifugation to remove impurities, so as to obtain a pure silk fibroin solution. Solidify the silk fibroin solution at a temperature of 66°C to obtain the required silk fibroin glasses blank. After visible spectrum analysis, the light transmittance of this glasses blank is 96%; and the hydrophilic property is good, and the contact with water The angle is 22.3°.

Example 4

First, we remove the silkworm pupae from the cocoons, mince the cocoons and put them in a flask, use NaHCO ₃ solution as a degumming agent to soak at 25°C for 18 hours, and control its solubility to 0.5%. It is necessary to remove sericin at a temperature of 78°C 0.5h. Then the obtained silk fibers were washed with deionized water, and dried at a temperature of 53°C. Mince the silk fibroin fibers again and put them into the flask, add CaCl ₂ -H ₂ O solution with a solubility of 42% as a solvent, soak for 16 hours at 25°C, and dissolve the silk fibroin fibers at a temperature of 72°C. After cooling for 0.8 hours, put the silk fibroin solution in a cellulose separation membrane and dialyze for 4 days to remove small molecules in the solvent, and then remove impurities through high-speed centrifugation to obtain a pure silk fibroin solution. Solidify the silk fibroin solution at a temperature of 45°C to obtain the required silk fibroin glasses blank. After visible spectrum analysis, the light transmittance of this glasses blank is 95%; and the hydrophilic property is good, and the contact with water The angle is 21.8°C.

Claims (5)

1. the extracting method of a silk fibroin, carry out according to the following steps::

1, the silkworm chrysalis in the removal silk cocoon, it is standby that silk cocoon is cleaned rubbing;

2, use Na ₂CO ₃, NaHCO ₃In one or both as degumming agent, its solubility is controlled at 0.2-6%, comes unstuck the fibroin fiber that obtains under temperature 70-100 ℃;

3, fibroin fiber is placed in the deionized water soaks, then 30-100 ℃ of oven dry down;

4, silk fibroin is dissolved in the CaCl that solubility is 35-60% ₂-H ₂O or CaCl ₂-C ₂H ₆O-H ₂In the O solution, temperature is 60-80 ℃;

5, silk fibroin protein solution is contained in dialysis, centrifugation in the separation membrane made from cellulose, obtains the pure solution of silk fibroin.

2. according to the extracting method of claim 1, it is characterized in that: in step 2, described degumming agent solubility is 0.4-0.8%.

3. according to the extracting method of claim 1 or 2, it is characterized in that: before step 2 is come unstuck, will soak 8-20 hour down at 20-60 ℃ through the silk cocoon that step 1 is handled.

4. according to the extracting method of claim 1 or 2, it is characterized in that: in step 4, CaCl ₂-H ₂O or CaCl ₂-C ₂H ₆O-H ₂O solution solubility is 40-45%.

5. according to the extracting method of claim 1 or 2, it is characterized in that: before step 4 dissolving, will soak 8-20 hour down at 20-50 ℃ through the fibroin fiber that step 3 is handled.