CN114557907A

CN114557907A - Application of phycocyanin nanoparticles in preparation of sunscreen preparation

Info

Publication number: CN114557907A
Application number: CN202210246316.3A
Authority: CN
Inventors: 邬诗媛; 曹进; 郑明雪; 李国艳; 旦增桑姆; 丹增曲尼; 戚雪勇
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2022-05-31

Abstract

The invention discloses an application of phycocyanin nanoparticles in preparing a sunscreen preparation. Firstly, phycocyanin freeze-dried powder is extracted from marine algae spirulina and sphingomyelina; then dissolving the phycocyanin freeze-dried powder in ultrapure water, and adjusting the pH to 8.3-8.5 by using a NaOH solution; slowly dripping ethanol under stirring, then adding glutaraldehyde, and stirring to promote crosslinking of the nanoparticles; centrifuging, removing supernatant, washing the nanoparticles with absolute ethanol to remove redundant phycocyanin and glutaraldehyde, finally adding ultrapure water and ultrasonically dissolving the nanoparticles in water to obtain successfully prepared phycocyanin nanoparticles. The obtained phycocyanin nanoparticles are used for developing photo-protection active ingredients to prepare the sun-screening gel, and the sun-screening gel has the sun-screening effect of resisting UV.

Description

Application of nano-particles of phycocyanin in preparation of sunscreen preparation

Technical Field

The invention belongs to the field of protein construction, and particularly relates to application of a phycocyanin nano-preparation in a sun-screening preparation, and particularly relates to application of phycocyanin nano-particles in preparation of a sun-screening gel.

Background

Ultraviolet (UV) is one of the most common factors affecting human health and disease. UV includes UVA (320-400nm), UVB (280-320nm) and UVC (200-280 nm). UVA and UVB cause loss of collagen and problems of spots, aging and the like after reaching the earth surface, and UVC reaches the earth surface in a small amount and causes little damage to the skin. It can destroy human DNA, resulting in DNA damage, damaged DNA replication, gene mutation and further influence the synthesis and function of specific protein. Therefore, there is a market demand for novel sunscreen agents having excellent ultraviolet absorption, strong radical scavenging ability, strong adhesive ability, and no cytotoxicity.

Common sunscreen agents on the market are physical sunscreen agents, chemical sunscreen agents and bioengineering sunscreen agents. The physical sunscreen agent mainly comprises TiO₂And ZnO, and can form a protective film on the skin surface to reflect ultraviolet rays. However, physical sunscreens still have certain disadvantages, such as inorganic TiO₂And ZnO has a molecular diameter of about 100nm and readily penetrates into the stratum corneum of the epithelial layer (PC), and recent studies have shown that TiO is easily penetrated into the stratum corneum of the epithelial layer (PC)₂It also has some cytotoxicity. The chemical sunscreen is an aromatic compound capable of absorbing ultraviolet rays, generates resonance quanta through molecular resonance, and converts the light energy of the ultraviolet rays into visible light to play a role in protection. However, chemical sunscreens are liable to enter blood, and are decomposed into radicals or intermediates harmful to human bodies under the action of light, so that oxidative deterioration is liable to occur, and skin allergy is caused. In addition to this, the present invention is,

the engineering sunscreen agents mainly play a role by regulating the immune function of the skin and increasing the activity of cells, and few reports are reported at present. Through literature research, the sunscreen component with the photoprotective effect is generally found to have excellent characteristics of ultraviolet absorption, free radical scavenging capacity, adhesion capacity and no cytotoxicity.

Phycocyanin (PC) is a natural light-harvesting protein extracted from seaweed, is a water-soluble macromolecular protein with chromophore, is difficult to permeate into skin or even circulate like small molecules, and has good biocompatibility and higher safety. The PC can emit strong fluorescence, the fluorescence does not disappear due to the existence of other biomolecules, the PC has good light absorption performance and a high absorption coefficient in a wide spectral range, is a potential photoprotective active ingredient, has the potential of chemical sun protection, can prevent photoaging, can be used as a natural anti-inflammatory agent, and reduces the possibility of activation of skin inflammatory cytokines. In addition, PC can also remove free radicals, prevent DNA oxidative damage, and improve skin immunity. In addition, after the phycocyanin is prepared into the nanoparticles through the cross-linking agent, the nanoparticles have the characteristic of physical sun-screening particles. The literature of using the sunscreen composition to prepare sunscreen products has not been reported.

Disclosure of Invention

In order to overcome the defects of various physical and chemical photo-protection active substances on the market at present, the invention utilizes the characteristics of phycocyanin and the advantages that phycocyanin nanoparticles have chemical and physical sun-screening agents at the same time, and applies the phycocyanin nanoparticles to the development of photo-protection and sun-screening products.

The invention constructs a protein nanoparticle-phycocyanin nanoparticle and prepares the protein nanoparticle-phycocyanin nanoparticle into a sun-screening gel with good sun-screening effect, the bioengineering sun-screening gel not only has excellent ultraviolet absorption effect and no cytotoxicity, but also has strong free radical scavenging capacity and adhesion capacity, can overcome a plurality of defects of sun-screening products in the current market, and can meet certain market demands.

An application of nano-particles of phycocyanin in preparing the sun-screening medicine is disclosed.

An application of nano-particles of phycocyanin in preparing sun-screening gel is disclosed.

A sun-screening gel prepared from phycocyanin nanoparticles comprises the following components in parts by weight:

wherein the concentration of the phycocyanin nanoparticle heavy suspension is 10 mg/mL. The nanoparticle resuspension of phycocyanin is prepared by the following steps:

(1) extracting phycocyanin lyophilized powder from Spirulina and Sphingomonas of marine algae;

(2) dissolving phycocyanin lyophilized powder in ultrapure water, and adjusting pH to 8.3-8.5 with NaOH solution; obtaining phycocyanin solution;

(3) under stirring, dripping ethanol into the phycocyanin solution obtained in the step (2) at a certain dripping speed, then adding glutaraldehyde, and stirring to promote the crosslinking of the nano particles;

(4) centrifuging the product obtained in the step (3), removing the supernatant, washing the nanoparticles for several times by using absolute ethyl alcohol to remove redundant phycocyanin and glutaraldehyde, finally adding ultrapure water and ultrasonically dissolving the nanoparticles in water to obtain the phycocyanin nanoparticle heavy suspension.

In the step (2), the dosage proportion of the phycocyanin freeze-dried powder to the ultrapure water is 10 mg: 1 mL; the concentration of the NaOH solution was 0.1M.

In the step (3), the volume ratio of the phycocyanin solution to the ethanol to the glutaraldehyde is 2: 8: 0.2; the volume percentage concentration of the glutaraldehyde is 1 percent; the dropping speed of the ethanol is 1 min/mL; the stirring speed is 390-450r/min, and the stirring time is 4-6 h.

In the step (4), the centrifugation speed is 10000-.

The preparation method of the sunscreen gel comprises the following steps:

(1) firstly, adding 3/4 of the total amount of purified water into carbomer 940, and preparing a 4% aqueous solution after full swelling;

(2) adding 1/8 (the total amount of the propylene glycol and the purified water) into the aqueous solution obtained in the step (1), uniformly mixing, and adding triethanolamine under stirring to form a gel matrix;

(3) and (3) adding the phycocyanin nanoparticle heavy suspension into the gel matrix in the step (2) under continuous stirring, adding the rest purified water to full volume, and stirring uniformly to obtain the phycocyanin nanoparticle sun-screening gel.

The invention has the beneficial effects that:

(1) phycocyanin is composed of a carrier group and a linearly extended tetrapyrrole compound with a chromophore open; the carrier group and the chromophore are linked by a thioether linkage; the fluorescent phycocyanin PC can emit strong fluorescence, the fluorescence does not disappear due to the existence of other biomolecules, the fluorescent phycocyanin PC has good light absorption performance and a high absorption coefficient in a wide spectrum range, and is a potential photo-protection active component; has good photo-protection effect, and can effectively resist ultraviolet rays when being used as a photo-protection active component in a sun-screening preparation.

(2) The phycocyanin nanoparticles are water-soluble macromolecular proteins with chromophores, are difficult to permeate into skin or even circulate like small molecules, have high safety, have anti-inflammatory and antioxidant capabilities, prevent photoaging, can be used as a natural anti-inflammatory agent, and reduce the possibility of activation of skin inflammatory cytokines. Compared with phycocyanin PC, the ultraviolet-proof fabric can reflect partial ultraviolet rays, realizes physical sun protection and has better sun protection performance.

(3) The invention overcomes the defects of various physical and chemical photo-protection active substances in the market at present, and the obtained sunscreen gel has excellent ultraviolet absorption effect, no cytotoxicity, strong oxidation resistance, strong free radical scavenging capacity and strong adhesion capacity, can meet certain market requirements, and has good application value and social benefit.

Drawings

FIG. 1 is a diagram of the diameter of phycocyanin nanoparticles DLS.

FIG. 2 is a TEM morphology of phycocyanin nanoparticles.

The left and right images of fig. 3 are respectively the ultraviolet full-wavelength scanning spectra of phycocyanin solution and phycocyanin nanoparticles.

FIG. 4 shows emission spectra of phycocyanin nanoparticles excited at 285nm and 325 nm.

FIG. 5 phycocyanin nanoparticle resuspension.

FIG. 6 phycocyanin nanoparticle sunscreen gel.

FIG. 7 shows UV absorbance values of two sunscreen gels (wherein PC represents phycocyanin solution; PCNP represents phycocyanin nanoparticles).

Detailed Description

The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

Example 1

(1) The preparation of the phycocyanin nanoparticle resuspension adopts a glutaraldehyde crosslinking method.

Firstly, 20mg of phycocyanin freeze-dried powder is dissolved in 1mL of ultrapure water, and the pH value is adjusted to 8.3-8.5 by using NaOH (0.1M) solution. Slowly dropping 8mL of ethanol into the phycocyanin solution at the speed of 1min/mL under stirring (390 plus 450r/min), then adding 200 mu L of 1% glutaraldehyde, stirring for 4-6h to promote the crosslinking of the nanoparticles, centrifuging at 10000 plus 13000rpm for 15-20 min, removing the supernatant after centrifuging, washing the nanoparticles for three times by using absolute ethyl alcohol to remove redundant phycocyanin and glutaraldehyde, finally adding ultrapure water and ultrasonically promoting the dissolution of the nanoparticles into water to obtain the phycocyanin nanoparticle heavy suspension.

As shown in fig. 5, the phycocyanin nanoparticle resuspension was blue in color.

(2) A sunscreen gel was prepared by the following steps:

A. firstly, 0.5g of carbomer 940 is added into 12mL of purified water, and a 4% aqueous solution is prepared after full swelling;

B. adding 3g of propylene glycol and 2mL of purified water into the aqueous solution obtained in the step (1), uniformly mixing, and adding 0.5g of triethanolamine under stirring to form a gel matrix;

C. and (3) adding 1g of phycocyanin nanoparticle heavy suspension (10mg/mL) into the gel matrix in the step (2) under continuous stirring, adding the rest 2mL of purified water, and uniformly stirring to obtain the phycocyanin nanoparticle sun-screening gel.

Example 2: determination of phycocyanin nanoparticle particle size and TEM

The diameter of the phycocyanin nanoparticle DLS prepared by the method is about 280nm, and the dispersion coefficient PDI is about 0.2, which is shown in figure 1.

And adding ultrapure water into 100 mu L of the resuspended phycocyanin nanoparticle solution to 1mL, shaking uniformly, dripping one drop of the phycocyanin nanoparticle solution on a copper net, airing, dripping one drop of the phycocyanin nanoparticle solution, and observing the morphology of the nanoparticles by using a transmission electron microscope after dripping three times.

As shown in a TEM image of FIG. 2, the material has a spherical morphology, is uniformly dispersed and has an average particle size of about 40 nm. The large DLS particle size of the phycocyanin nanoparticles is probably caused by the fact that the surface of the phycocyanin nanoparticles is coated with a hydrated film when the phycocyanin nanoparticles are dispersed in water.

Example 3: spectral properties of phycocyanin nanoparticles

(1) Ultraviolet absorption spectrum of phycocyanin nanoparticles

Diluting the resuspended phycocyanin nanoparticles into a solution with a proper concentration, preparing the phycocyanin solution with the same concentration, respectively carrying out full-wavelength scanning on the two obtained solutions within the wavelength range of 200-800nm, and recording the absorption spectrum.

The experimental results are shown in fig. 3, the phycocyanin nanoparticles and the phycocyanin solution prepared by the method have good ultraviolet absorption at 285nm and 325nm, which indicates that both have the characteristics of photoprotective activity.

(2) Fluorescence spectrum of phycocyanin nanoparticles

Diluting the prepared phycocyanin nanoparticle solution, detecting the fluorescence spectrum of the phycocyanin nanoparticles by using a fluorescence spectrophotometer of Shimadzu Japan, and respectively selecting 285nm and 325nm as fluorescence excitation wavelengths, wherein the scanning range of the emission wavelength is 400nm-800 nm.

The experimental result is shown in fig. 4, the emission wavelengths obtained at 285nm and 325nm are both larger than 550nm, which indicates that the phycocyanin nanoparticle has the fluorescence property of releasing larger than 400nm and no harm to human body, and the nano preparation has safer skin light protection performance.

Example 4: preparation comparison of phycocyanin nanoparticles (PCNP) and Phycocyanin (PC) solutions sunscreen gel:

the matrix material is usually swollen in a solvent to prepare a gel matrix, and then the drug solution and other additives are added. The water-soluble medicine can be dissolved in water or glycerol, the water-insoluble medicine powder is ground with the water or the glycerol and then mixed with the gel matrix, and finally, the quantification is carried out, and the uniform stirring is carried out. In this example, the preparation steps of the sunscreen gel include the following:

(1) firstly, adding carbomer 940 into purified water, and preparing into 4% aqueous solution after fully swelling;

(2) adding propylene glycol and a proper amount of purified water into the fully swollen 4% aqueous solution, uniformly mixing, and adding triethanolamine under stirring to form a gel matrix;

(3) and (3) adding the phycocyanin nanoparticle heavy suspension/phycocyanin solution into the gel matrix in the step (2) under continuous stirring, adding the residual purified water to full volume, and stirring uniformly to obtain the phycocyanin nanoparticle sun-screening gel.

The sunscreen gel prepared by the method is light blue gel as shown in fig. 6.

Example 5: experiment for evaluating sun-proof effect by ultraviolet spectrophotometer method

According to the ultraviolet absorbent and shielding in the sunscreen cosmetic, the sunscreen cosmetic can not only block ultraviolet property, but also be coated on a breathable adhesive tape, artificial skin or a special substrate, ultraviolet irradiation with different wavelengths is carried out, the absorbance or ultraviolet absorption curve of a sample is measured by using an ultraviolet spectrophotometer method, and the sunscreen effect is directly balanced according to the size of test paper. The invention relates to an experiment for evaluating the sun-proof effect by using an ultraviolet spectrophotometer method, which comprises the following steps:

(1) cutting 3M adhesive tape into 1cm × 4cm, adhering on the light-transmitting side surface of quartz cuvette, connecting with power supply of ultraviolet/visible spectrophotometer, preheating instrument, and setting wavelength of UVB region to 285nm, 290nm, 295nm, 300nm, 305nm, 310nm, 315nm, and 320 nm;

(2) placing the quartz cuvette pasted with the adhesive tape in a sample light path and a reference light path, and adjusting the zero point of an instrument;

(3) accurately weighing 6mg of a sample to be detected, uniformly coating the sample on a quartz cuvette with a 3m adhesive tape, placing the cuvette in a drying oven at 35 ℃, and drying for 30 min;

(4) placing a sample cuvette to be measured in a sample light path, placing another quartz cuvette attached with an adhesive tape in a reference light path, respectively measuring ultraviolet absorbance values of set wavelengths in a UVB region, and then taking an arithmetic mean value of each measured value;

(5) and sequentially measuring five parallel samples, measuring the mean value of the five samples according to the method, and calculating the arithmetic mean value of the mean values of the five samples to obtain the absorbance of the test sample.

And (3) respectively carrying out ultraviolet absorbance detection on the phycocyanin nanoparticle (PCNP) sunscreen gel and Phycocyanin (PC) solution sunscreen gel samples according to the experimental operation steps, recording the mean value of the samples, and obtaining a detection result shown in figure 7.

And (3) evaluation of test results:

(1) if the absorbance value is less than 1.0 +/-0.1, the sample has no sun-proof effect.

(2) If the absorbance value is 1.0 ± 0.1, it indicates that the sample has a low sunscreen effect, and is suitable for winter, spring, autumn, morning and evening, and rainy days.

(3) If the absorbance value is greater than 1.0 + -0.1 and less than 2.0 + -0.2, it indicates that the sample has a moderate sunscreen effect and is suitable for moderate intensity sunlight irradiation.

(4) If the absorbance value is more than 2.0, the sample has a high-grade sun-proof effect and is suitable for sun irradiation in summer or outdoor activities, traveling and the like.

The experimental results are compared with the test result evaluation, so that the phycocyanin nanoparticle sun-blocking gel sample has a medium-grade sun-blocking effect, is suitable for medium-intensity sunlight irradiation, and the sun-blocking effect is superior to that of a phycocyanin solution sun-blocking gel sample, which shows that the phycocyanin nanoparticle sun-blocking gel has more excellent light protection characteristics.

Claims

1. An application of nano-particles of phycocyanin in preparing the sun-screening medicine is disclosed.

2. An application of nano-particles of phycocyanin in preparing sun-screening gel is disclosed.

3. A sunscreen gel prepared from phycocyanin nanoparticles is characterized in that the sunscreen gel comprises the following components in parts by weight:

4. the sunscreen gel of claim 3, wherein the concentration of the phycocyanin nanoparticle resuspension is 10 mg/mL.

5. The sunscreen gel of claim 3, wherein the nanoparticle resuspension of phycocyanin is prepared by:

6. The sunscreen gel of claim 5, wherein in step (2), the ratio of the lyophilized phycocyanin powder to the ultrapure water is 10 mg: 1 mL; the concentration of the NaOH solution was 0.1M.

7. The sunscreen gel of claim 5, wherein in step (3), the volume ratio of phycocyanin solution, ethanol, and glutaraldehyde is 2: 8: 0.2; the volume percentage concentration of the glutaraldehyde is 1 percent; the dropping speed of the ethanol is 1 min/mL; the stirring speed is 390-450r/min, and the stirring time is 4-6 h.

8. The sunscreen gel of claim 5 wherein in step (4), the centrifugation speed is 10000-13000rpm and the centrifugation time is 15-20 minutes.

9. A process for the preparation of a sunscreen gel as claimed in claim 3 comprising the steps of:

(2) adding propylene glycol and purified water into the aqueous solution obtained in the step (1), uniformly mixing, and adding triethanolamine under stirring to form a gel matrix;

10. The method of claim 9, wherein in step (1), the amount of purified water added is 3/4; in the step (2), the adding amount of the purified water is 1/8 of the total amount of the purified water; in the step (3), the adding amount of the purified water is 1/8 of the total amount of the purified water.