Disclosure of Invention
Aiming at the prior art, the invention aims to provide a nonionic facial cleanser with sterilizing and inflammation diminishing functions and a preparation method thereof. The facial cleanser disclosed by the invention is added with defensin components, so that the skin with thin, damaged and dark yellow epidermis can be quickly repaired. Can effectively inhibit bacteria, eliminate red swelling, eliminate skin toxin, dredge capillary vessels, simultaneously soften cutin of epidermis, balance oil secretion, shrink pores, and is beneficial to clearing blackheads and acnes. Enhancing skin resistance, and preventing scar hyperplasia. Meanwhile, the skin care product has the effects of mildly sterilizing, diminishing inflammation, regulating sebum secretion, promoting the growth of epidermal cells and enabling the damaged skin to heal without pigment and scars. Regulating skin pH, balancing oil and fat, killing bacteria, relieving inflammation, shrinking pores, and preventing wrinkle and acne.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided the use of a defensin in the preparation of a facial cleanser.
Preferably, the amino acid sequence of the defensin is: CYCRIPACIAGERRYGTCIYGGRLWAFCC, respectively; (SEQ ID NO.1)
More preferably, the defensin comprises one or more D-type amino acids in the amino acid sequence; specifically, the amino acid sequence is as follows:
CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC;
wherein X1 is Ala or D-Ala;
x2 is Thr or D-Thr;
x3 is Gly or D-Gly;
x4 is Phe or D-Phe;
wherein at least one of X1, X2, X3, or X4 is a D amino acid.
The invention provides a nonionic facial cleanser with sterilizing and inflammation diminishing functions, which comprises the following components in percentage by weight:
2.0-4.0% of beeswax, 6.0-8.0% of paraffin, 0.4-0.8% of defensin, 8.0-12.0% of vaseline, 30-35% of white oil, 3.0-5.0% of lanolin alcohol, 1.5-2.5% of glyceryl monostearate, 603.0-5.0% of tween-603, 7.0-9.0% of glycerol, 1-3% of polygonatum extract, 2-4% of lithospermum extract and the balance of deionized water.
Preferably, the polygonatum odoratum extract is prepared by the following method:
drying rhizoma Polygonati Odorati, pulverizing, sieving with 40-80 mesh sieve, and supercritical CO 2 The extraction process is carried out under the extraction pressure of 25-35MPa, the extraction temperature of 30-40 ℃ and CO 2 The flow rate is 25-28L/h, the entrainer is 95% ethanol, the amount of the entrainer is 4-6% of the weight of the raw materials, and the extraction time is 1-2h, so as to obtain the polygonatum odoratum extract.
Preferably, the lithospermum extract is prepared by the following method:
pulverizing radix Arnebiae, adding 70-80% ethanol 4-6 times of radix Arnebiae, ultrasonic extracting for 20-40min for 2-4 times, filtering, mixing filtrates, and recovering ethanol under reduced pressure until the filtrate has no alcohol smell to obtain radix Arnebiae extract.
In a third aspect of the present invention, a preparation method of the nonionic facial cleanser with bactericidal and anti-inflammatory effects is provided, which comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 70-80 deg.C, and stirring for 10-20 min to obtain mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain mixed solution C, cooling the mixed solution C to 30-40 deg.C, adding defensin, rhizoma Polygonati Odorati extract and radix Arnebiae extract, stirring for 5-10 min, and homogenizing for 5-10 min.
Preferably, in the step (1), the step (2) and the step (3), the stirring speed is 40-60 r/min.
The invention has the beneficial effects that:
(1) the facial cleanser disclosed by the invention takes defensins as a main bactericide, has high antibacterial property and extremely strong bacteriostatic and bactericidal effects on drug-resistant bacteria; and has no toxic and side effects, no residue and no bacterial drug resistance.
(2) The facial cleanser provided by the invention has multiple functions of cleaning, moistening, skin care, whitening and the like while sterilizing and diminishing inflammation.
(3) The components in the facial cleanser have a synergistic effect, can sterilize and diminish inflammation, can repair skin multiply, and is safe and non-irritant; and the preparation method is simple, can be used for industrial production, and is beneficial to popularization and application.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background section, defensins have broad-spectrum antimicrobial activity and remarkable bactericidal effect, and are widely applied in the fields of plant disease-resistant genetic engineering, biopharmaceuticals and the like. However, the stability, hydrolysis resistance and bioactivity of defensins still need to be further improved, and therefore, structural modification of defensins becomes a hot point of research.
The introduction of non-natural D-amino acid is one of the methods for modifying the structure of defensin, and the stability, hydrolysis resistance and biological activity of defensin can be improved by properly introducing D-amino acid. However, the type, position and amount of the introduced D-amino acid are critical to the structural modification effect of the defensin, and if the type, position or amount of the introduced D-amino acid is not appropriate, the stability, hydrolysis resistance and biological activity of the defensin are reduced.
In the present invention, the amino acid composition of the defensin used is: CYCRIPACIAGERRYGTCIYGGRLWAFCC, respectively; (SEQ ID NO. 1).
On the basis, the invention firstly carries out structural modification on the defensin, and a large number of experiments show that the defensin has better effect when modified according to the following amino acid composition:
CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC;
wherein X1 is Ala or D-Ala;
x2 is Thr or D-Thr;
x3 is Gly or D-Gly;
x4 is Phe or D-Phe;
wherein at least one of X1, X2, X3, or X4 is a D-form amino acid.
The defensins can be prepared by conventional methods in the prior art such as a chemical synthesis method or a genetic engineering method.
The method for modifying the structure of the defensin can directly mix D-type amino acid in chemical synthesis; or D-amino acid is added into the culture medium in the process of gene engineering expression.
On the basis of carrying out structural modification research on the defensins, the defensins are used for preparing the facial cleanser for the first time. In one embodiment of the invention, the facial cleanser is prepared from the following raw materials:
2.0-4.0% of beeswax, 6.0-8.0% of paraffin, 0.4-0.8% of defensin, 8.0-12.0% of vaseline, 30-35% of white oil, 3.0-5.0% of lanolin alcohol, 1.5-2.5% of glyceryl monostearate, 603.0-5.0% of tween-603, 7.0-9.0% of glycerol, 1-3% of polygonatum extract, 2-4% of lithospermum extract and the balance of deionized water.
In the facial cleanser, the raw material components are an organic whole, and the raw materials have a synergistic promoting effect. The radix polygonati officinalis extract and the lithospermum erythrorhizon extract are compounded, and a synergistic promoting effect can be generated between the radix polygonati officinalis extract and the defensin, so that the sterilization effect is improved. The bactericide is compounded with additional components such as a surfactant, a humectant, an emollient and the like, and the optimized additional components not only can enhance the stability of the bactericide and facilitate the preservation, but also can obviously enhance the bactericidal activity.
In the test process, raw material components such as defensins, the polygonatum extract and the lithospermum extract are removed in sequence to prepare the facial cleanser. Then, the prepared facial cleanser is subjected to a bacteriostatic test, and the result shows that compared with the facial cleanser disclosed by the invention, the bacteriostatic effect of the facial cleanser is reduced by deleting the raw material components.
In conclusion, the facial cleanser is prepared by optimizing the selection and the dosage of the raw materials. The facial cleanser disclosed by the invention has multiple functions while sterilizing and diminishing inflammation: can quickly repair the skin with thin, damaged and dark yellow epidermis. Can effectively inhibit bacteria, eliminate red swelling, eliminate skin toxin, dredge capillary vessels, simultaneously soften cutin of epidermis, balance oil secretion, shrink pores, and is beneficial to clearing blackheads and acnes. Enhancing skin resistance, and preventing scar hyperplasia. Meanwhile, the skin care product has the effects of mildly sterilizing, diminishing inflammation, regulating sebum secretion, promoting the growth of epidermal cells and enabling the damaged skin to heal without pigment and scars. Regulating skin pH, balancing oil and fat, killing bacteria, relieving inflammation, shrinking pores, and preventing wrinkle and acne.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples and comparative examples of the present invention are conventional in the art and are commercially available.
Example 1: preparation of facial cleanser
1. The raw materials comprise (by weight percent):
3.0% of beeswax, 7.0% of paraffin, 0.6% of defensin, 10.0% of vaseline, 32% of white oil, 4.0% of lanolin alcohol, 2.0% of glyceryl monostearate, tween-604.0, 8.0% of glycerol, 2% of polygonatum extract, 3% of lithospermum extract and the balance of deionized water.
Wherein the amino acid sequence of the defensin is as follows:
CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC;
wherein X1 is D-Ala; x2 is D-Thr; x3 is D-Gly; x4 is D-Phe.
The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.
The polygonatum extract is prepared by the following method:
drying rhizoma Polygonati Odorati, pulverizing, sieving with 40-80 mesh sieve, and supercritical CO 2 The extraction process is carried out, the extraction pressure is 30MPa, the extraction temperature is 35 ℃, and CO is used 2 The flow rate is 26L/h, the entrainer is ethanol with the volume concentration of 95%, the use amount of the entrainer is 5% of the weight of the raw materials, and the extraction time is 1.5h, so that the polygonatum extract is obtained.
Preferably, the lithospermum extract is prepared by the following method:
pulverizing radix Arnebiae, adding 75% ethanol 5 times the weight of radix Arnebiae, ultrasonic extracting for 3 times (30 min each time), filtering, mixing filtrates, and recovering ethanol under reduced pressure until the filtrate has no alcohol smell to obtain radix Arnebiae extract.
2. The preparation method comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain a mixed solution C, cooling the mixed solution C to 35 ℃, adding the defensin, the polygonatum extract and the lithospermum extract, stirring for 10 minutes, and then homogenizing for 5 minutes to obtain the traditional Chinese medicine composition.
Example 2: preparation of facial cleanser
1. The raw materials comprise (by weight percent):
2.0% of beeswax, 8.0% of paraffin, 0.4% of defensin, 12.0% of vaseline, 30% of white oil, 5.0% of lanolin alcohol, 1.5% of glyceryl monostearate, tween-605.0, 7.0% of glycerol, 3% of polygonatum extract, 2% of lithospermum extract and the balance of deionized water.
Wherein the amino acid sequence of the defensin is as follows:
CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC;
wherein, X1 is Ala; x2 is D-Thr; x3 is Gly; x4 is D-Phe.
The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.
The preparation method of the polygonatum odoratum extract is the same as that of example 1.
The preparation method of the lithospermum extract is the same as that of the example 1.
2. The preparation method comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 70 deg.C, and stirring for 20 min to obtain mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain a mixed solution C, cooling the mixed solution C to 30 ℃, adding the defensin, the polygonatum extract and the lithospermum extract, stirring for 10 minutes, and homogenizing for 10 minutes to obtain the traditional Chinese medicine composition.
Example 3: preparation of facial cleanser
1. The raw materials comprise (by weight percent):
4.0% of beeswax, 6.0% of paraffin, 0.8% of defensin, 8.0% of vaseline, 35% of white oil, 3.0% of lanolin alcohol, 2.5% of glyceryl monostearate, tween-603.0%, 9.0% of glycerol, 1% of polygonatum extract, 4% of lithospermum extract and the balance of deionized water.
Wherein the amino acid sequence of the defensin is as follows:
CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC;
wherein, X1 is Ala; x2 is D-Thr; x3 is Gly; x4 is Phe.
The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.
The preparation method of the polygonatum odoratum extract is the same as that of example 1.
The preparation method of the lithospermum extract is the same as that of the example 1.
2. The preparation method comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 80 deg.C, and stirring for 10 min to obtain mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain a mixed solution C, adding the defensin, the polygonatum extract and the lithospermum extract when the mixed solution C is cooled to 40 ℃, stirring for 5 minutes, and then homogenizing for 5 minutes to obtain the traditional Chinese medicine composition.
Comparative example 1: preparation of facial cleanser
1. The raw materials comprise (by weight percent):
3.0% of beeswax, 7.0% of paraffin, 10.0% of vaseline, 32% of white oil, 4.0% of lanolin alcohol, 2.0% of glyceryl monostearate, tween-604.0, 8.0% of glycerol, 3% of lithospermum extract and the balance of deionized water.
Wherein, the preparation of the lithospermum extract is the same as that of example 1.
2. The preparation method comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain a mixed solution C, adding the lithospermum extract when the mixed solution C is cooled to 35 ℃, stirring for 10 minutes, and then homogenizing for 5 minutes to obtain the lithospermum-lithospermum composite material.
Comparative example 2:
1. the raw materials comprise (by weight percent):
3.0% of beeswax, 7.0% of paraffin, 10.0% of vaseline, 32% of white oil, 4.0% of lanolin alcohol, 2.0% of glyceryl monostearate, tween-604.0, 8.0% of glycerol, 2% of polygonatum odoratum extract and the balance of deionized water.
Wherein, the preparation of the polygonatum extract is the same as that of example 1.
2. The preparation method comprises the following steps:
(1) mixing beeswax, paraffin, vaseline, lanolin alcohol and white oil, heating to 90 deg.C, and stirring to obtain mixed solution A;
(2) adding glyceryl monostearate, tween-60, glycerol and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution B;
(3) adding the mixed solution A into the mixed solution B to obtain a mixed solution C, adding the polygonatum extract when the mixed solution C is cooled to 35 ℃, stirring for 10 minutes, and then homogenizing for 5 minutes to obtain the polygonatum-odoratum tea beverage.
And (3) safety test:
the facial cleansers prepared in examples 1 to 3 were tested according to the microbiological examination method, toxicological test method and human safety examination method in technical standards for cosmetic safety (2015 edition), and the results all meet the requirements of the standards, indicating that the facial cleanser of the present invention is safe to use.
Test example 1: bacteriostasis test
1. Test materials: examples 1 to 3 and comparative examples 1 to 2.
2. Preparation of test bacteria:
respectively inoculating Escherichia coli, Staphylococcus aureus and Candida albicans into the beef extract culture solution in a sterile operating room, and culturing at 28-30 deg.C for 1-3 d.
3. The test method comprises the following steps:
adopting an agar punching diffusion method, dividing a sterile culture dish into 6 groups, wherein each group comprises 5, pouring the sterilized beef extract culture solution into the sterile culture dish, each dish is 15-20 mL, sucking 0.1mL of test bacteria such as escherichia coli, staphylococcus aureus and candida albicans to the culture dish by using a sterile pipette after the beef extract culture solution is solidified, adding the test bacteria to the culture dish, uniformly coating, and adding different strains into each culture dish of each group.
After the coating, 6 holes were punched in the medium at equal intervals using a sterilized stainless steel punch with an outer diameter of 4 mm. mu.L of the facial cleansers prepared in example 1, example 2, example 3, comparative example 1 and comparative example 2 were each pipetted with a micro-sampler, and an equal amount of sterile water was added to one well of the sample to prepare a control group, which was then labeled. The bacteria were incubated at 37 ℃ for 24h, and the molds and yeasts were incubated at 28 ℃ for 72 h. And measuring the diameter (measured by the bacteriostatic diameter mm) of the bacteriostatic zone, repeating the test for 5 times, and taking the average value of 5 times as the diameter of the bacteriostatic zone.
4. And (3) test results:
the test results are shown in table 1.
Table 1: data of bacteriostatic test
As can be seen from the table 1, the facial cleanser prepared by the invention has obvious inhibiting effect on escherichia coli, staphylococcus aureus and candida albicans; in the facial cleansers prepared in comparative examples 1-2, the defensin, the polygonatum extract and the lithospermum extract are absent, so that the inhibition effect on the bacteria is remarkably reduced, and the defensin, the polygonatum extract and the lithospermum extract can synergistically enhance the antibacterial effect.
The facial cleanser prepared in the embodiment 1 of the invention is stored in an incubator (relative humidity is more than 75%) at 37 ℃ for three months, and then the bacteriostasis test is carried out according to the method. The result shows that the bacteriostatic activity of the facial cleanser is not obviously reduced after the facial cleanser is stored for three months. The stability of the facial cleanser of the invention is better.
Test example 2: fibroblast proliferation assay
1. Test samples: examples 1 to 3 and comparative examples 1 to 2.
The above test samples were dried to obtain powders, respectively, and then prepared into test sample solutions with a concentration of 0.5mg/ml using PBS.
2. The test method comprises the following steps:
preparing the prepared suspension of human fibroblasts (obtained from the residual normal skin after operation, generation 4-10) into a suspension with the concentration of 2.5 multiplied by 10 4 Inoculating each/ml of the cells into a 96-well plate, adding culture medium with corresponding volume without inoculating cells in a blank control group, adding 20 mu l of PBS into each well of a negative control group after the cells are attached to the wall, and adding 20 mu l of test into each well of a sample groupSample solutions (prepared in PBS) were prepared, each set of 6 duplicate wells, and placed in an incubator (37 ℃ C., 5% CO) 2 ) And then added with human MTT (purchased from Sigma, lot number: 08797HJ) solution (in PBS) 20. mu.l, continued in CO 2 Incubator (37 ℃, 5% CO) 2 ) And (4) incubating. The 96-well plate was removed, the medium in each well carefully aspirated, DMSO added, and mixed well. And measuring the absorbance value at 570nm by using an enzyme-linked immunosorbent assay detector, and comparing with a negative control group to obtain the relative proliferation rate of the sample group.
In the formula: t-sample well Abs; c-negative control well Abs; c0-blank control well Abs.
The test results are shown in Table 2.
Table 2: fibroblast proliferation assay results
Test sample
|
Fibroblast proliferation Rate (%)
|
Example 1
|
24.8
|
Example 2
|
20.6
|
Example 3
|
18.3
|
Comparative example 1
|
10.2
|
Comparative example 2
|
8.6 |
The change of the structure of the dermis is the main cause of skin aging, and fibroblasts are the main cells constituting the dermis layer. The number of fibroblasts in the dermis layer gradually decreases as aging occurs and the skin thickness gradually thins. Therefore, the promotion of fibroblast proliferation is an important way to solve the problem of skin aging. As can be seen from Table 2, the facial cleanser of the present invention has the synergistic effect of promoting the proliferation of human fibroblasts, confirming that the facial cleanser of the present invention has the anti-aging effect.
Test example 3: test for inhibiting melanogenesis
1. Test samples:
the facial cleansers prepared in example 1 and comparative examples 1 to 2.
2. The test method comprises the following steps:
human skin melanoma cells SK-MEL-1 were placed in minimal essential medium containing 10% bovine embryo serum and incubated at 37 ℃ and 5% CO 2 Culturing under the conditions of (1). The cultured cells were smeared on the bottom of a 75-well flask, and the number of cells was 3X 10 5 Well, left overnight, allowing the cells to adhere to the wall. After confirmation, each medium was changed to a new medium containing 10ppm of the test sample. The control group used DMSO (dimethyl sulfoxide) -containing medium.
In the above manner, each medium was changed to a new medium containing the test sample once every 2 days until the cultured cells were sufficient to fill the flask. Then, the medium fluid was taken out, washed with PBS (phosphate buffered saline), and dissolved in 1N NaOH, and the Optical Density (OD) at 500 nm was measured, and the inhibition rate of melanin formation was calculated according to the following formula 1, and the results are shown in table 3.
Formula 1: melanin formation inhibition (%) {1- (OD of each experimental sample/OD of control group) } × 100.
Table 3: results of the test for inhibiting melanogenesis
Test sample
|
Melanin formation inhibition ratio (%)
|
Example 1
|
48.2
|
Comparative example 1
|
18.4
|
Comparative example 2
|
12.0 |
As can be seen from table 3, the facial cleanser of the present invention significantly inhibited the formation of melanin and exhibited a whitening effect on the skin, as compared to comparative examples 1 and 2.
Test example 4: evaluation on trial
The facial cleansers prepared in examples 1 to 3 and comparative examples 1 to 2 were used as samples for trial evaluation, which mainly reflected in evaluation of foam, cleanliness and moistening feeling during the trial process.
The specific method comprises the following steps: the samples were distributed to 20 test persons for comparison. The test persons used the samples and washed in the same way, each sample was used for two weeks, and the test persons scored according to their own feelings. The score was taken as 10 points, 1 point being the worst and 10 points being the best.
The specific cleaning method of the tester comprises the following steps: the face cream is used in the morning and evening every day, firstly, the face skin is moistened by clear water, then, a proper amount of sample is taken and added into the palm, and after being kneaded and foamed, the face cream is smeared on the face and the periphery of eyes and the face skin is gently softened for about one minute; then washing the mixture by using clear water; the above procedure was repeated every morning and evening, and after two weeks of use of each sample, the product was scored and the results are shown in table 4.
Table 4: trial evaluation results
Test items
|
Foaming speed
|
Richness of foam
|
Cleanliness after washing
|
Degree of moistening after washing
|
Example 1
|
9.5
|
9.5
|
1.0
|
9.8
|
Example 2
|
9.2
|
9.0
|
9.4
|
9.0
|
Example 3
|
9.0
|
9.0
|
9.2
|
8.8
|
Comparative example 1
|
8.4
|
9.0
|
8.0
|
7.8
|
Comparative example 2
|
8.8
|
9.0
|
8.2
|
7.4 |
As can be seen from table 4, compared with the samples prepared in comparative examples 1 and 2, the facial cleanser prepared in the examples of the present invention has improved foaming speed, foam richness, cleanliness after washing and moistening after washing during the trial process.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Jihai Biotechnology Ltd of Rong Cheng City
<120> nonionic bactericidal anti-inflammatory facial cleanser and preparation method thereof
<130> 2019
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> PRT
<213> defensins
<400> 1
Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr Gly
1 5 10 15
Thr Cys Ile Tyr Gly Gly Arg Leu Trp Ala Phe Cys Cys
20 25