CN114344345B

CN114344345B - Seaweed extract capable of effectively relieving skin sunburn and preparation method and application thereof

Info

Publication number: CN114344345B
Application number: CN202210021156.2A
Authority: CN
Inventors: 陈华; 孙恢礼; 潘剑宇; 蔡冰娜; 万鹏; 陈得科
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2022-01-10
Filing date: 2022-01-10
Publication date: 2022-07-26
Anticipated expiration: 2042-01-10
Also published as: CN114344345A

Abstract

The invention discloses a seaweed extract capable of effectively relieving skin sunburn and a preparation method and application thereof. The preparation method comprises the following steps: weighing a certain amount of seaweed, adding water with the volume of 20-50 times, leaching with boiling water for 0.5-4 h, cooling the leaching solution, centrifuging for 10-15 min by 3000-10000 g, sequentially carrying out suction filtration and ultrafiltration on the centrifuged supernatant, collecting the ultrafiltration trapped fluid, concentrating and freeze-drying to obtain the seaweed extract. The preparation method can remove the components which do not obviously improve the activity of ultraviolet irradiation cells, and highly enrich the active components which effectively relieve the sunburn of the skin in the seaweed extract, thereby improving the physiological effect of the seaweed extract and greatly reducing the dosage of the seaweed extract. The seaweed extract has high safety, can obviously reduce apoptosis and necrosis caused by ultraviolet irradiation, reduce inflammatory exudation of dermis and degradation of elastic fiber, and effectively relieve skin sunburn caused by ultraviolet rays.

Description

Seaweed extract capable of effectively relieving skin sunburn and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a seaweed extract capable of effectively relieving skin sunburn and a preparation method and application thereof.

Background

With the ozone layer destroyed, the skin is exposed to ultraviolet rays in excess for a long time, resulting in an increasing incidence of photodermatosis, basal cell carcinoma, squamous cell carcinoma, and the like. After the skin is over-irradiated by ultraviolet rays in sunlight, local acute phototoxic dermatitis is also often generated, and polymorphous light eruptions, herpes simplex, solar urticaria, erythema multiforme, porphyria cutanea tarda, lupus erythematosus, vitiligo, telangiectasia, insolation and the like are also easily excited. Therefore, it is very important to find various measures to effectively reduce the occurrence of skin damage and canceration caused by ultraviolet irradiation.

The external-applied sunscreen cosmetic can effectively prevent skin photodamage and reduce skin cancer. The sunscreen agent in the sunscreen cosmetic mostly contains benzene rings and unsaturated side chains, and adverse reactions such as allergic contact dermatitis and the like occur after ultraviolet irradiation, so that the wide application of the sunscreen agent is limited to a certain extent. Many clinically usable drugs are for preventing and treating skin photoaging, such as retinoids, isotretinoin, tazarotene, retinol, antioxidants (vitamin C, vitamin E, β -carotene, coenzyme Q10, superoxide dismutase SOD, etc.), depigmenting agents (hydroquinone, arbutin, etc.), keratolytic agents (α -hydroxy acid, β -hydroxy acid, etc.), cytokines (epidermal growth factor EGF, fibroblast growth factor FGF, platelet-derived growth factor PDGF, etc.), natural biological extracts (epigallocatechin gallate EGCG, anthocyanin, etc.), trace elements (selenium), sex hormones, etc., and relatively few agents and drugs for protecting skin from sunburn.

The marine habitat is unique, and rich biological resources are stored, so that the marine habitat is a resource treasure house for searching precious medicines. The sargassum fusiforme is a brown algae plant, is widely distributed in China along the sea, and not only has rich nutritive value, but also has remarkable medical effect. The medical functions of the sargassum fusiforme are recorded in medical works such as Shennong herbal classic and the like in ancient China, and the sargassum fusiforme is one of a few marine traditional Chinese medicinal materials recorded in past editions of Chinese pharmacopoeia and has exact curative effect. The pharmacopoeia determines its functions and indications, namely hardness softening and resolving, phlegm eliminating, diuresis inducing, goiter, scrofula, testicular tumor, phlegm-fluid retention and edema. The detection and analysis show that the sargassum fusiforme contains rich polysaccharide components, dietary fibers, multiple vitamins, 17 amino acids, mineral substances and trace elements, and has multiple effects of preventing and resisting cancers, improving the immunity of the organism, reducing blood pressure, preventing arteriosclerosis and thrombosis, reducing blood sugar and the like.

Disclosure of Invention

The invention aims to provide a seaweed extract which can effectively prevent skin sunburn and promote sunburn repair. The seaweed extract has good skin compatibility, can remarkably improve the activity of epidermal cells after ultraviolet irradiation, reduce apoptosis and necrosis, reduce dermal inflammatory infiltration and elastic fiber degradation, and relieve skin sunburn.

Another object of the present invention is to provide a method for preparing an extract of seaweed by leaching with boiling water, centrifugation, ultrafiltration separation. The method is simple, low in cost, green, environment-friendly, short in production period and suitable for industrial production.

Still another object of the present invention is to provide a use of the seaweed extract. The seaweed extract can be used in medicine in the form of lotion, ointment, tincture, liniment, spirit, powder, oil, cataplasm, plaster, plastics, aerosol, etc.; the seaweed extract can also be used in cosmetics, and the cosmetic forms can include cream, lotion, gel, jelly, aqua, spray, etc.; the seaweed extract can also be used in biomedical materials, such as lotions, solutions, dressings, ointments, etc., and the types of dressings can include films, hydrocolloids, hydrogels, sponges, sprays, etc.

The invention realizes the aim through the following technical scheme:

in one aspect, the present invention provides a seaweed extract having good water solubility and skin compatibility.

The above seaweed extract is prepared by extracting seaweed with boiling water, centrifuging, ultrafiltering, concentrating, and lyophilizing.

The seaweed can include but is not limited to one or a combination of several of undaria pinnatifida, kelp, sargassum fusiforme, gulfweed, sargassum pallidum and carrageen. Preferably, the seaweed is derived from sargassum fusiforme.

The seaweed can be sun-dried or oven-dried seaweed, or fresh seaweed.

As an alternative embodiment, the preparation method of the seaweed extract comprises the following steps: weighing a certain amount of seaweed, adding water with the mass volume ratio of 20-50 times g/ml, leaching with boiling water for 0.5-4 h, cooling the leaching solution, centrifuging for 10-15 min by 3000 g-10000 g, sequentially performing suction filtration and ultrafiltration with a 3k-5kDa ultrafiltration membrane on the centrifuged supernatant, collecting ultrafiltration trapped fluid, concentrating and freeze-drying to obtain the seaweed extract.

Preferably, the centrifugal supernatant is filtered by suction filtration, the filtrate is filtered by an ultrafiltration membrane with the molecular weight cutoff of 5kDa, and the retentate (>5kDa) is collected, concentrated and freeze-dried.

Preferably, the centrifugal supernatant is filtered by an ultrafiltration membrane with the aperture of 0.45 mu m and the cut-off molecular weight of 3kDa in sequence, and the cut-off solution (3 kDa-0.45 mu m) is collected, concentrated and freeze-dried.

Preferably, the sargassum fusiforme is weighed, water with the mass volume ratio of 25 times g/ml is added, the mixture is extracted for 1 hour by boiling water, the extract liquid is cooled and then centrifuged for 10 minutes by 10000g, the centrifuged supernatant liquid is sequentially filtered by suction and ultrafiltered by a 5kDa ultrafiltration membrane, the ultrafiltration trapped fluid is collected, concentrated and freeze-dried, and the sargassum fusiforme is obtained.

In another aspect, the invention provides an application of a seaweed extract with a significant skin sunburn protection effect.

Because the skin irradiated by ultraviolet rays has obvious difference in physiological state and structure from the skin not irradiated by ultraviolet rays, no substance which is reported to be used for ordinary skin care at present can also be directly used for the care of the skin irradiated by ultraviolet rays. Based on the understanding of the mechanism of action of skin damage caused by ultraviolet irradiation, the process and mechanism of repair of sunburn of skin, and the like, the present inventors prefer the seaweed extract obtained by leaching with boiling water to be used for the care of ultraviolet-irradiated skin.

As an alternative embodiment, the seaweed extract may be used for preventing sunburn of the skin. The final concentration of administration is preferably 50. mu.g/mL-1600. mu.g/mL. More preferably 100. mu.g/mL-800. mu.g/mL.

As an alternative embodiment, the seaweed extract may also be used for the treatment of sunburn.

In a preferred embodiment of the present invention, the seaweed extract is used in a pharmaceutical product; preferably, the seaweed extract is particularly suitable for use in a medicament effective for repairing sunburn of the skin, and the medicament may be in the form of a lotion, an ointment, a tincture, a liniment, spirit, a powder, an oil, a cataplasm, a plaster, a film coating agent, an aerosol, etc.

When used in medicine, the seaweed extract accounts for 0.01-0.5% of the medicine by mass.

In a preferred embodiment of the present invention, the seaweed extract may be used in cosmetics; preferably, the seaweed extract is particularly suitable for use in cosmetics effective for preventing sunburn of the skin, and the cosmetic dosage forms include creams, lotions, gels, mists, and the like.

When the seaweed extract is used in cosmetics, the mass fraction of the seaweed extract in the cosmetics is 0.005-0.5%. In a preferred embodiment, the seaweed extract is present in the cosmetic in an amount of 0.02 to 0.5% by weight.

When used in cosmetics, the seaweed extract may be formulated with a cosmetic base and may also be formulated with a sunscreen agent, which refers to a UVA protective agent and/or a UVB protective agent.

The UVA protective agent is preferably one or a combination of more of disodium salt of 2,2 '-bis- (1, 4-phenylene) 1H-benzimidazole-4, 6-disulfonic acid, methylene bis-benzotriazolyl tetramethyl butyl phenol, p-xylylene dicamphor sulfonic acid and bis-ethylhexyloxyphenol methoxyphenyl triazine, and the using amount of the UVA protective agent is that the disodium salt of 2, 2' -bis- (1, 4-phenylene) 1H-benzimidazole-4, 6-disulfonic acid is 0.1-4.5%, the methylene bis-benzotriazolyl tetramethyl butyl phenol is 0.1-3.5%, the p-xylylene dicamphor sulfonic acid is 0.1-4.5%, and the bis-ethylhexyloxyphenol methoxyphenyl triazine is 0.2-4.5% by weight of the total weight of the cosmetic.

The UVB protective agent is preferably phenyl benzimidazole sulfonic acid and potassium, sodium and triethanolamine salts thereof, ethylhexyl methoxycinnamate, cresyl trazol trisiloxane, TiO ₂ One or a combination of several of the above components, the dosage of which is, according to the percentage of the total mass of the cosmetic, 0.1 to 3 percent of phenyl benzimidazole sulfonic acid and potassium, sodium and triethanolamine salts thereof, 0.02 to 8 percent of ethylhexyl methoxycinnamate, 0.2 to 8 percent of cresol trozole trisiloxane, TiO ₂ 0.5％～6％。

The above UVA protective agents, UVB protective agents and cosmetic bases are commercially available.

In a preferred embodiment of the invention, the seaweed extract may also be used in biomedical materials; preferably, the seaweed extract is particularly suitable for biomedical materials which can effectively repair skin sunburn, the dosage forms of the biomedical materials comprise lotion, solution, dressing, ointment and the like, and the types of the dressing comprise film, hydrocolloid, hydrogel, sponge, spray and the like.

When the seaweed extract is used in biomedical materials, the mass fraction of the seaweed extract in the biomedical materials is 0.005-0.5%. In a preferred embodiment, the seaweed extract is present in the biomedical material in an amount of 0.02 to 0.5% by weight.

It is worth mentioning that the seaweed extract provided by the invention is used as a functional component, which can not cause skin allergy and photosensitive reaction, and can also obviously reduce the active oxygen level in epidermal cells irradiated by ultraviolet rays, stabilize the intracellular calcium ion concentration and mitochondrial membrane potential, improve cell cycle retardation and reduce the apoptosis and necrosis of the epidermal cells. The seaweed extract can also remarkably improve the activity of antioxidase such as CAT and the like in ultraviolet irradiation skin, greatly reduce the oxidation damage products of DNA such as 8-OHdG and the like in skin tissues, obviously relieve the symptoms such as epidermis thickening, dermal inflammatory infiltration, collagen degradation and the like, and has the function of remarkably relieving skin sunburn.

Therefore, when the seaweed extract of the present invention is applied to cosmetics or biomedical materials, the choice of the base ingredients of cosmetics, the type, amount, preparation process, etc. of raw materials for biomedical materials is wide. All ingredients used in cosmetic and biomedical materials should be dermatologically acceptable and not interfere with the properties of the original seaweed extract of the present invention, i.e., not cause undue toxicity, incompatibility, instability, allergic response, and the like when in contact with human skin or when compatible with other ingredients.

The solution of the invention is based on the understanding of the inventor on the skin sunburn and repair mechanism, the dose-effect relationship of the seaweed extract, the skin photosensitivity and the like, and combined with the research results of modern cosmetics, biomedical materials and pharmacology, the seaweed extract which has good compatibility with the skin, no sensitization and photosensitivity and obvious skin sunburn prevention and treatment effects is searched, excavated and prepared through a large number of creative experiments.

The invention has the following beneficial effects:

(1) firstly, the seaweed extract is dug to have the effect of preventing and treating the skin sunburn. Sargassum fusiforme is preferably selected from a plurality of seaweeds such as undaria pinnatifida, kelp, sargassum fusiforme, sargassum pallidum, caraway and carrageen, and is prepared by using boiling water extraction, centrifugation, suction filtration and ultrafiltration separation technologies to obtain the seaweed extract. The seaweed extract can rapidly infiltrate into skin surface layer, and has multiple effects of preventing sunburn, relieving inflammation, and repairing sunburn. The seaweed extract can remarkably improve the natural barrier function and the ultraviolet resistance of the skin and effectively relieve the sunburn of the skin at a lower dose (50 mu g/mL).

The invention relates to a seaweed extract prepared by 8 methods of normal-temperature water extraction, boiling water extraction, dilute alkali extraction, dilute acid extraction, ultrasonic treatment, cellulase enzymolysis, pectinase enzymolysis and helicase enzymolysis, although the seaweed extract prepared by boiling water extraction is not optimal in vitro antioxidant activity, the seaweed extract has the most excellent performance in the aspects of improving the activity of ultraviolet irradiation cells and relieving the skin sunburn of mice caused by ultraviolet irradiation, and is obviously better than other 7 preparation methods.

(2) The seaweed extract has the advantages of simple preparation process, low cost, environmental protection and short production period, is suitable for large-scale industrial production, and has great application prospect. The algae boiling water leaching liquor is processed by an ultrafiltration membrane with the molecular weight cutoff of 5kDa or 3kDa, and components (containing salts, free amino acids, fatty acids and the like) which do not obviously improve the activity of ultraviolet irradiation cells can be removed, so that the obtained algae extract has better physiological effect and less discharged pollutants.

In the examples of the present invention, comparative experiments were performed, and compared with the seaweed extract YS obtained in example 3, the two components (precipitate and supernatant) of the seaweed extract prepared by ethanol precipitation treatment did not significantly improve the effect of alleviating cell damage caused by ultraviolet irradiation, but rather reduced the efficacy thereof, indicating that the whole composition of the seaweed extract YS as precipitate and supernatant can exert a better effect.

The invention also compares the filtrate before and after ultrafiltration with the trapped liquid of the ultrafiltration membrane of the 3k-5kDa ultrafiltration membrane, finds that the obtained seaweed extract (trapped liquid component) has the best effect of relieving cell damage caused by ultraviolet irradiation, and has the sun-screening effect which is obviously superior to the seaweed extract prepared without ultrafiltration treatment and the seaweed extract of the ultrafiltration filtered liquid component.

Thus, the seaweed extract of the present invention obtained unexpected effects.

(3) The seaweed extract has good water solubility and skin compatibility, does not cause skin irritation, is not sensitized, has no photosensitivity and remarkable effect, and can be widely applied to cosmetics and biomedical materials. By adopting the ultrafiltration separation technology, the seaweed extract can be highly enriched, the dosage of the seaweed extract is greatly reduced while the physiological effect is improved, and the requirements on the stability, the compatibility and the like of the seaweed extract in the preparation process of cosmetics and biomedical materials and the operation difficulty are further effectively reduced.

Drawings

FIG. 1 is a graph showing the effect of the algal extract in example 4 on HaCaT cell activity (YS 1: the algal extract prepared in example 1; YS 2: the algal extract prepared in example 2; YS 3: the algal extract prepared in example 3).

FIG. 2 is a graph showing the effect of the seaweed extract of example 5 on the activity of UVB-irradiated HaCaT cells (YS 1: the seaweed extract prepared in example 1; YS 2: the seaweed extract prepared in example 2; YS 3: the seaweed extract prepared in example 3); (in contrast to the UVB model group, ^* P<0.05， ^** P<0.01)。

FIG. 3 is a graph of the effect of seaweed extract on the survival status of UVB-irradiated HaCaT cells in example 6 (Control: blank Control; UVB: UVB model; UVB + YS-L: 200. mu.g/mL seaweed extract administered after UVB modeling; UVB + YS-H: 800. mu.g/mL seaweed extract administered after UVB modeling); (Calcein-AM: fluorescence shows live cells; PI: fluorescence shows dead cells; Merged: fluorescence shows cell live/dead states).

FIG. 4 is a photograph (100X) of HE staining of mouse skin in example 7 (Control: blank Control; UVB: UVB model; UVB + YS 1: algal extract prepared in example 1 was administered after UVB molding; UVB + YS 2: algal extract prepared in example 2 was administered after UVB molding; UVB + YS 3: algal extract prepared in example 3 was administered after UVB molding).

FIG. 5 is a photograph of HE staining of mouse skin (100X) in example 9 (Control: blank Control; UVB: UVB model; UVB + Placebo: gel base applied after UVB molding; UVB + SFP: seaweed extract gel applied after UVB molding).

FIG. 6 is a measurement of the content of 8-OHdG in skin tissue in example 9 (Control: blank; Model: UVB Model; Placebo: UVB Model followed by gel matrix; SFP: UVB Model followed by seaweed extract gel); ( ^* P<0.05， ^** P<0.01; in contrast to the Control, ^# P<0.05)。

FIG. 7 is the SOD enzyme activity assay in skin tissue in example 9 (Control: blank Control; Model: UVB Model; Placebo: UVB gel matrix applied after molding; SFP: UVB gel applied after molding); ( ^* P<0.05， ^** P<0.01; in contrast to the Control, the Control is, ^## P<0.01)。

FIG. 8 is a KEGG pathway enrichment analysis (day 7) of significantly differentially expressed mRNA in transcriptome sequencing SFP-administered group compared to UVB model group in example 9.

FIG. 9 is an evaluation of the antioxidant activity of the algal extract prepared in comparative example 1 (DPPH radial scavenging effect: DPPH radical scavenging activity; superoxide anion scavenging activity; ABTS radial scavenging effect: ABTS radical scavenging activity; ferritic reducing antioxidant power: ferric ion reducing antioxidant ability; hydroxyradical scavenging activity; linoleic acid peroxidation inhibiting activity); (RM water: normal temperature water extraction; skinning water: boiling water extraction; NaOH: dilute alkali extraction; HCl: dilute acid extraction; ultrasonic wave: ultrasonic treatment; cellulose: cellulase enzymolysis; pectase: pectinase enzymolysis; snailase: snailase enzymolysis); (in contrast to the building water group, ^* P<0.05， ^** P<0.01)。

FIG. 10 is a graph of the effect of the seaweed extract prepared in comparative example 1 on the activity of UVB irradiated HaCaT cells (Control: blank Control; Model: UVB Model; RM water: aqueous extraction at room temperature; building water: extraction with boiling water; NaOH: aqueous alkali; HCl: dilute acid; ultrasonic wave; cellulose: enzymatic hydrolysis; pectase: pectinase enzymatic hydrolysis; snailase: helicase enzymatic hydrolysis); ( ^** P<0.01)。

FIG. 11 is a graph showing the effect of the seaweed extract prepared in comparative example 2 on HaCaT cell Activity ^** P<0.01)。

FIG. 12 is the effect of seaweed extract prepared in comparative example 2 on the Activity of UVB-irradiated HaCaT cells (( ^* P<0.05， ^** P<0.01; in comparison with the UVB model group, ^## P<0.01)。

FIG. 13 is a graph showing the effect of seaweed extract prepared in comparative example 3 on the activity of UVB-irradiated HaCaT cells (ii) ^* P<0.05， ^** P<0.01; in comparison with the UVB model group, ^# P<0.05， ^## P<0.01)。

Detailed Description

The invention is further described with reference to the accompanying drawings, but the embodiments of the invention are not limited to the following examples, and equivalent variations or modifications of the method according to the invention are to be considered within the scope of the invention. The starting materials used below are all commercially available, unless otherwise indicated.

In one embodiment of the present invention, the seaweed extract is mixed with physiological saline to prepare a solution (aqua) and applied to the skin by wet application. The results showed that, compared to the control product without seaweed extract (UVB model group, gauze applied with physiological saline only), the gauze applied with seaweed extract was significantly reduced in skin damage degree 2 days after uv irradiation, and only showed epidermal hypertrophy, hyperkeratosis, and dermal inflammatory cell infiltration (superficial or middle necrosis of epidermal and dermal tissues, numerous collagen fiber breaks, disorganization, epithelial necrosis of hair follicle and sebaceous gland, and full inflammatory cell infiltration in UVB model group); on the 5 th day after the ultraviolet irradiation, the inflammatory cell number is recovered to be normal (the UVB model group still has the phenomena of epidermal thickening, hyperkeratosis, incrustation, acanthocyte hyperplasia and inflammatory cell infiltration of dermis and subcutaneous tissues); on day 7 after the ultraviolet irradiation, the skin of the mice has no obvious difference with that of a blank control group (the UVB model group still can see thickening of the epidermis, the number of the cells of the dermis layer inflammation is reduced, and the number of the new-born hair follicles is obviously increased).

In the preferred embodiment of the invention, the seaweed extract is taken as a functional component, and can be matched with a conventional cosmetic matrix to prepare a cosmetic as an experimental sample; the control sample was a cosmetic base without seaweed extract. The results showed that the skin epidermal thickness of mice treated with the experimental sample containing the seaweed extract was reduced by 35.89% (about 11.7 μm) and the fibroblast cell number was increased by 65.00% (about 19.5/field) compared to the control sample.

In the preferred embodiment of the invention, the seaweed extract is used as a functional component, and a conventional biomedical material matrix can be matched to prepare a biomedical material as an experimental sample; the control sample was a biomedical material matrix without seaweed extract. The results showed that the skin of mice treated with the test sample containing the seaweed extract was significantly reduced in the degree of damage at day 5 after the UV irradiation (no significant difference from the blank control group), and that the epidermis was free from inflammatory exudation, desquamation, incrustation, etc., as compared with the control sample.

To further illustrate the solution of the present invention and facilitate its use, the following examples take the preparation of solutions (aqueous), emulsions, gels, supplemented with cosmetic or biomedical matrices formulated with seaweed extracts. It should be recognized by those of skill in the art that the components contained in these examples are not intended as limitations on the practice of the inventive concepts, but are merely an enabling alternative.

The seaweed extract adopted by the invention is homogenized or mixed with any of a plurality of cosmetic or biomedical material matrix components by the conventional process, and the cosmetic or biomedical material with obvious skin sunburn protection effect can be prepared. For example, the substances except the seaweed extract in example 8 are replaced conventionally to prepare other types or the same type of cosmetics; similar benefits would be obtained by making other types or the same types of biomedical materials, except that the seaweed extract of example 9 is routinely replaced.

Example 1

Taking edible sargassum fusiforme sun-dried alga meeting the use standard of related marine products in China, cleaning for 3-4 times by using distilled water, drying and crushing to obtain dry powder. Weighing 200g of dry powder, adding 10L of distilled water, and boiling with 100 ℃ water for 0.5 h. After the leach liquor was cooled, 3000g were centrifuged for 15 min. And (4) carrying out suction filtration on the centrifugal supernatant (filtration qualitative filter paper), filtering the filtered solution by using an ultrafiltration membrane with the molecular weight cutoff of 5kDa, collecting ultrafiltration cutoff solution, concentrating and freeze-drying to obtain the seaweed extract.

Researches show that the seaweed extract prepared by the embodiment has good biocompatibility and no sensitization, does not cause skin photosensitive reaction, can obviously improve apoptosis and necrosis caused by ultraviolet irradiation, reduces inflammatory exudation of dermis and degradation of collagen and elastic fibers after the ultraviolet irradiation, reduces adverse reactions of erythema, inflammation, effusion, desquamation, scab and the like caused by the skin after the ultraviolet irradiation with large dose, and effectively relieves sunburn of the skin.

Example 2

Weighing 200g of edible fresh sargassum fusiforme meeting the use standard of related marine products in China, washing with deionized water for 3-4 times, crushing, adding 4L of deionized water, and carrying out boiling water bath at 100 ℃ for 4 hours. After cooling, 8000g of the suspension were centrifuged for 12 min. The supernatant was sequentially filtered through an ultrafiltration membrane with a pore size of 0.45 μm and a molecular weight cut-off of 3 kDa. Collecting the trapped fluid with the diameter of 3 kDa-0.45 mu m, concentrating and freeze-drying to obtain the seaweed extract.

Researches show that the seaweed extract prepared by the embodiment has good biocompatibility and no sensitization, does not cause skin photosensitive reaction, can obviously improve apoptosis and necrosis caused by ultraviolet irradiation, reduce inflammatory exudation of dermis and degradation of collagen and elastic fibers after the ultraviolet irradiation, relieve adverse reactions of erythema, inflammation, exudate, desquamation, scabbing and the like caused by the skin after the ultraviolet irradiation with large dose, and effectively relieve the skin sunburn.

Example 3

Weighing 200g of edible sargassum fusiforme sun-dried algae meeting the use standard of related marine products in China, soaking in distilled water for 30min, cleaning for 3-4 times, and soaking in distilled water for 30min again. Mincing Sargassum fusiforme, adding 5L distilled water, and boiling with 100 deg.C water for 1 hr. After cooling the leaching liquor, centrifuging for 10min at 10000 g. The supernatant was filtered by suction (qualitative filter paper), and the filtrate was filtered through an ultrafiltration membrane having a molecular weight cut-off of 5 kDa. Collecting the ultrafiltration retentate, concentrating, and lyophilizing to obtain Sargassum extract.

Example 4

The influence of the seaweed extract prepared by the invention on the cell activity was investigated.

The cell activity was measured by the CCK-8 method: will have a density of 6X 10 ⁴ one/mL of HaCaT cell suspension was inoculated into a 96-well plate (100. mu.L/well), and the plate was incubated at 37 ℃ with 5% CO ₂ Cultured in an incubatorAnd (5) 24 h. mu.L of each well was aspirated, and 50. mu.L/well of a sample group was added with a sample solution (

final concentration

50, 100, 200, 400, 800, 1600. mu.g/mL, sterilized by a 0.45 μm pore size syringe filter) of the seaweed extract (prepared in examples 1-3) at the corresponding concentration. The blank control group was prepared by adding 50. mu.L/well of DMEM complete medium, respectively. Each group is placed in CO ₂ After further culturing in the incubator for 24 hours, the cell morphology was observed using a fluorescence inverted phase contrast microscope CKX41 (Olympus, Japan). Subsequently, 10. mu.L of CCK-8 solution was added to each well, and the incubation was continued for 4 hours, and absorbance was measured for each well at a measurement wavelength of 450nm and a reference wavelength of 650nm for each set of 6 parallel wells using a microplate reader Multiskan GO (Thermo Fisher Scientific Co.).

It was found that HaCaT cell activity of the seaweed extract-treated group was increased with the increase of the dose of the seaweed extract prepared according to the present invention (FIG. 1). The seaweed extract prepared by the invention has good biocompatibility and no obvious cytotoxicity.

Example 5

The influence of the seaweed extract prepared according to the present invention on the activity of UVB-irradiated cells was investigated.

UVB irradiation HaCaT cell damage modeling: using a skin photoaging tester HOPE-MED 8140A (Tianjin development area, general industry and trade Co., Ltd.), a UVB-313EL lamp tube (wavelength 290-315 nm), irradiation dose 20mJ/cm ² 。

The cell activity was measured by the CCK-8 method: the density is 6 multiplied by 10 ⁴ one/mL of HaCaT cell suspension was inoculated into a 96-well plate (100. mu.L/well), and the plate was incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator for 24 h. mu.L of the cell damage model group (negative control group) was aspirated and 50. mu.L of each well was used to wrap the culture plate with tinfoil, and the cell damage model groups (UVB + sample group, UVB model group) were mixed at 20mJ/cm ² UVB irradiation was performed at doses. After the end of UVB irradiation, 50. mu.L/well of a sample solution (

final concentration

50, 100, 200, 400, 800, 1600. mu.g/mL, sterilized by a 0.45 μm pore size syringe filter) of the corresponding concentration of the seaweed extract (prepared in examples 1, 2, 3) was added to the UVB + sample group. The negative control group and UVB model group were added with DMEM complete medium, 50. mu.L/well, respectively. Each group is placed in CO ₂ After further culturing in the incubator for 24 hours, the cell morphology was observed using a fluorescence inverted phase contrast microscope CKX41 (Olympus, Japan). Subsequently, 10. mu.L of CCK-8 solution was added to each well, the incubation was continued for 4 hours, and absorbance was measured for each well using a microplate reader Multiskan GO (Thermo Fisher Scientific Co.) at a measurement wavelength of 450nm and a reference wavelength of 650nm for each set of 6 parallel wells.

It was found that as the dosage of the seaweed extract prepared by the method of the invention is increased, the activity of HaCaT cells after UVB irradiation of the seaweed extract treatment group is also obviously improved, and the HaCaT cells have obvious difference compared with the UVB model group (figure 2). The seaweed extract prepared by the invention can obviously relieve the adverse effect of ultraviolet irradiation on the cell activity.

Example 6

The effect of the seaweed extract prepared according to the present invention on the state of the HaCaT cells irradiated with UVB was studied.

The experimental procedure of example 5 was followed to model the damage of HaCaT cells irradiated with UVB. The density is 0.9 multiplied by 10 ⁵ The cell suspension was inoculated into 6-well plates at 2 mL/well, incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator for 24 h. The medium was aspirated off, and fresh DMEM complete medium was added at 1.5 mL/well. Wrapping the non-UVB damage model group with tinfoil, placing into a skin photoaging tester together with the UVB damage model groups (UVB + sample group, UVB model group), measuring the ultraviolet intensity of the skin photoaging tester with an ultraviolet irradiation meter before ultraviolet irradiation, and converting to 20mJ/cm according to a formula ² Irradiation time corresponding to ultraviolet dose. After the irradiation, 500. mu.L of fresh DMEM complete medium (UVB model group) or sample medium (sterilized by a 0.22 μm needle filter) containing the seaweed extract (prepared in example 3, UVB + sample group) was added to each well, and CO was added ₂ The incubator continues to culture for 24 h. The culture medium was aspirated and washed 1 time with PBS, 2 mL/well. And absorbing PBS, adding 1mL Calcein AM detection working solution into each well, incubating for 30min in an incubator in a dark place, absorbing supernatant, incubating for 5min in a dark place with 1mL PI staining solution into each well, absorbing supernatant under a fluorescence microscope for observation, photographing, and calculating fluorescence intensity by using Image J.

The survival state of the HaCaT cells after UVB irradiation in the seaweed extract-treated group was significantly improved and was very significantly different from that in the UVB model group (fig. 3). The seaweed extract prepared by the invention can obviously improve the survival state of HaCaT cells after ultraviolet irradiation.

Example 7

The seaweed extracts prepared in examples 1-3 were evaluated for efficacy using an experimental model of skin damage in BALB/c mice irradiated with UVB.

The contents and procedures related to the animal experiment related to the experiment are in compliance with the relevant laws and regulations for the use and management of the experimental animals and the relevant regulations of the ethical committee of the experimental animals of the mechanism, and the welfare of the experimental animals is ensured.

65 SPF-grade BALB/c mice, 16-18 g of male mice, were purchased from Guangdong provincial medical laboratory animal center. Quarantine for 3 days, observing for 1 time every day, and removing unhealthy animals immediately. Grouping: blank control group (5), UVB model group (15), UVB + sample group (45 total, 15 per sample). Animal feeding conditions: 3 pieces per box, group culture. Raising temperature and humidity: 20-26 ℃, 40-70%, adopting a reaction time of 12 h: 12h day and night intermittent illumination; the conditions in the feeding room are always stable, and the mice can freely eat and drink water.

Depilation: day 1, after animal quarantine is qualified and grouped, the fur on the back of the animal is shaved off by a shaver, and the area is about 2 multiplied by 2cm ² . During the test, if the growth of the animal hair is found to affect the handling of the sample and observation, the shaving operation is also required. The depilation process should be gentle to avoid damaging the skin.

Administration to the samples: the animals began dosing on day 2 after depilation. In UVB + sample group, 0.8mL (1 mouse dose) of seaweed extract (1.6 mg/mL concentration, solvent was physiological saline) was taken and wetted area was about 2X 2cm ² The three layers of gauze were applied to the mouse skin after depilating, covered with cellophane, and then fixed with adhesive tape for 3 hours. After the completion of the operation, the adhesive tape, the cellophane and the gauze were removed, and the residual sample was washed with clean water. The animals in the placebo group and UVB model group were administered physiological saline according to the same procedure. The administration was 1 time per Day for 6 consecutive days (Day 2-Day 7).

Irradiating UVB: day 8, after the animals are dosed for 30min, the animals in the UVB model group and the UVB + sample group are subjected to intraperitoneal injection of sodium pentobarbital for anesthesia (the dose is 60mg/kg body weight), and after anesthesia, the animals are fixed in a skin photoaging tester HOPE-MED 8140A (Tianjin development area, general industry and trade Co., Ltd.) for UVB irradiation, wherein the UVB irradiation intensity is 0.174mW/cm ² The irradiation time was 28.7min, and the cumulative irradiation dose was 300 mJ. And gauze is used for covering the eyes of the mouse in the irradiation process, so that the eyes are prevented from being damaged by ultraviolet rays. After 30min of irradiation, the animals were dosed again as described above.

On

days

2, 5, and 7 after UVB irradiation (i.e., Day 10, Day 13, Day 15), 5 animals were taken for each of the UVB model group and UVB + sample group, and cervical dislocation was sacrificed. The skin of the back of the mouse after depilation, drug administration and irradiation is taken and fixed in neutral formaldehyde, and then paraffin section and HE staining are carried out for pathological examination.

According to research, compared with the UVB model group, the skin of the back of the mice in the alga extract treatment group prepared by the invention has obviously reduced damage degree on the 2 nd day after ultraviolet irradiation (superficial layer or middle layer necrosis of epidermis and dermis tissues in the UVB model group), the number of inflammatory cells returns to normal on the 5 th day (figure 4, spiny cell hyperplasia of the UVB model group and infiltration of dermis and subcutaneous tissue inflammatory cells), and the skin of the mice has no obvious difference with a blank control group on the 7 th day (the epidermis of the UVB model group is thickened and the number of dermal inflammatory cells is reduced).

The seaweed extract prepared by the invention can effectively relieve adverse reactions such as skin erythema, inflammatory infiltration, elastic fiber degradation, desquamation, scabbing and the like caused by ultraviolet irradiation.

Example 8

The seaweed extract obtained in the preparation of the example 3 is selected as a raw material to prepare the sunscreen functional cosmetic.

The ingredients for forming the cosmetic comprise, by mass fraction of 100%:

phase A: 0.5% of 4-methylbenzylidene camphor, 3% of fatty alcohol-polyoxyethylene ether, 9% of C12-15 benzoate (CAS #:68411-27-8), 2% of dioctyl carbonate, 0.8% of microcrystalline wax, 0.5% of hydrolyzed silk protein/PG-propyl methyl dihydroxy siloxane crosspolymer and 0.7% of VE acetate.

Phase B: 5% of 1, 3-butanediol, 4% of propylene glycol, 0.1% of EDTA-2Na, 3.5% of methylene bis-benzotriazolyl tetramethyl butyl phenol, 0.2% of PEG-40 hydrogenated castor oil, 0.8% of NaCl and the balance of deionized water.

And C phase: 0.5 percent of seaweed extract, 0.4 percent of allantoin and 0.3 percent of VC phosphate ester magnesium.

Phase D: 0.2 percent of essence and 0.1 percent of butyl paraben.

The process comprises the following steps: heating phase A and phase B to 80 deg.C respectively, adding phase A into phase B under stirring, and homogenizing for 10 min. Add phase C when cooling to 60 ℃. Cooling to 40 deg.C, adding phase D, and mixing to obtain cosmetic.

The prepared marine organism cosmetic (marked as A) and a control sample (marked as a, the seaweed extract is replaced by deionized water, and other matrix components and the using amount are the same as those of A) are subjected to efficacy evaluation.

40 BALB/c mice were taken and randomly divided into 4 groups: blank control group, UVB model group, UVB + marine organism cosmetics, UVB + control sample, 10/group. Dehairing and UVB molding were performed as in example 7. The administration mode (administration before UVB irradiation) was changed from wet application to application, and 200mg of cosmetic was administered per mouse. The blank control group and the UVB model group were not administered. Covering with cellophane, fixing with adhesive tape, applying for 3 hr, removing adhesive tape, cellophane and gauze, and cleaning with clear water for 1 time/day. On day 2 after UVB irradiation, mice were sacrificed by dislocation of cervical vertebrae, and skin on the back, which had been depilated, administered, irradiated, was fixed in neutral formaldehyde.

Placing the sealed sample under a 10-time lens to observe the integral shape of the skin, then taking a first picture from the uppermost end of the skin observed in a visual field under a 40-time lens, then taking one picture every 2mm after moving a scale, taking 5 pictures, then selecting 5 points on each picture by using a microscopic image analysis system, measuring the thickness of the epidermis of the picture, measuring 25 points in total on each picture, and taking the average value to be recorded as the thickness of the epidermis. The number of fibroblasts in each field was counted by taking 6 fields per picture, and the mean value was recorded as the number of fibroblasts in the dermis layer of each field (Table 1).

TABLE 1 Effect of Marine organism cosmetics on UVB irradiation of epidermal thickness and dermal fibroblasts in BALB/c mice

Note: in comparison with the blank control, the control, ^* P<0.05， ^** P<0.01; compared with the UVB model group, the method has the advantages that, ^# P<0.05， ^## P<0.01。

the study finds that the thickness of the epidermis of mice in the UVB model group and the UVB + control group is obviously increased, and the number of fibroblasts in the dermis layer is obviously reduced compared with the blank control group. Compared with the UVB model group and the UVB + control group, the thickness of the mouse epidermis in the UVB + marine organism cosmetic group is obviously reduced, the fibroblast number of the dermis layer is obviously improved, and the difference between the mouse epidermis thickness and the UVB + marine organism cosmetic group is not obvious.

The cosmetic prepared by the seaweed extract can effectively relieve adverse reactions such as dermal inflammatory infiltration, elastic fiber degradation, epidermal hyperkeratosis and the like caused by ultraviolet irradiation, and can be used for preventing skin sunburn.

Example 9

The seaweed extract obtained in example 3 was selected as a raw material to prepare sunburn repair gel (biomedical material).

The components of the seaweed extract gel comprise the following components in percentage by mass of 100 percent: 2% of medium viscosity sodium alginate (Mecline, CP, viscosity 200 + -20 mpa.s), 0.16% of seaweed extract, and the balance of deionized water. Firstly weighing the seaweed extract, adding deionized water, stirring uniformly, then adding sodium alginate, slowly dissolving and stirring uniformly to prepare gel.

Gel matrix: 2% medium viscosity sodium alginate solution (deionized water was used instead of equal amount of seaweed extract).

60 BALB/c mice were taken and randomly divided into 4 groups: blank control, UVB model, UVB + gel matrix, UVB + seaweed extract gel, 15/group.

Day 1, the animal's back coat was shaved with a shaver, and the area was about 2X 2cm ² The depilation process should be gentle,avoiding damaging the skin.

Day 2, in the skin depilatory part on the back of the mouse, the gel matrix is coated on the UVB + gel matrix group, the seaweed extract gel is coated on the UVB + seaweed extract gel group, and the dosage of 200mg of gel is administered to each mouse. The blank control group and the UVB model group were not administered.

The animals in the Day 3, UVB model group, UVB + gel matrix group and UVB + seaweed extract gel group were anesthetized by intraperitoneal injection of 3mg/mL sodium pentobarbital (injection volume: 20mL/kg body weight), and were fixed in a skin photoaging tester after anesthesia for a cumulative UVB irradiation dose of 300 mJ. And gauze is used for covering the eyes of the mouse in the irradiation process, so that the eyes are prevented from being damaged by ultraviolet rays. Animals in the placebo group were anesthetized with only sodium pentobarbital injection, but were not subjected to UVB irradiation. After the animals in the blank control group, the UVB model group, the UVB + gel matrix group and the UVB + seaweed extract gel group are subjected to ultraviolet irradiation for 30min, the administration mode is the same as the above. The administration was performed 1 time per day, and the animals were observed for survival, body shape, coat, skin, feces, muscle tone, gait, mental status, etc.

On

days

2, 5, and 14 after the irradiation with ultraviolet rays (Day 4, Day 7, and Day 16), 5 animals were taken from each group, and after shaving off the back hair, photographs were taken, and the frozen serum was examined for the 8-OHdG content with a kit. Meanwhile, skin tissues of a back molding and administration area are cut: fixing part of the gel in a neutral formaldehyde fixing solution, and then performing HE staining and Masson's trichrome staining respectively; after part of the liquid nitrogen is frozen quickly, the mixture is frozen and stored at the temperature of minus 80 ℃, and the activity of superoxide dismutase SOD is measured; after part of the liquid nitrogen is frozen quickly, the frozen product is frozen and stored at the temperature of minus 80 ℃ and then RNA-seq and RT-qPCR detection is carried out.

It was found that, compared with the UVB model group and the UVB + gel matrix group, the skin of the UVB + seaweed extract gel group mice was significantly reduced in the damage degree on the 5 th day (i.e., day 7) by uv irradiation, the inflammatory reaction and the collagen fiber degradation were less, the elastic fibers of the skin tissue were orderly arranged and partially maintained in a net-like interlaced structure, and the epidermis was not significantly different from the blank control group in inflammatory exudation, desquamation, scabbing, and the like (fig. 5).

In addition, compared with the UVB model group and the UVB + gel matrix group, the content of 8-OHdG (one of DNA oxidative damage products) in the skin tissue of the UVB + seaweed extract gel group mice is significantly reduced (fig. 6), the activities of antioxidant enzymes (SOD, CAT, and the like) are significantly improved (fig. 7), and adverse symptoms such as skin tissue redness and swelling are significantly improved.

The research shows that the seaweed extract can inhibit MAPK/AP-1 activation and NF-kB nuclear translocation (figure 8) by regulating Toll-like receptor pathways, chemokine signal pathways, cytokine-cytokine receptor interaction and other pathways, and can down-regulate the expressions of TNF, MMPs, IL-6, IL-1B and the like by adopting transcriptome analysis (RNA-seq) and RT-qPCR verification, so that the inflammatory reaction and collagen degradation are reduced, and the skin sunburn is effectively relieved.

Therefore, the seaweed extract prepared by the invention has no cytotoxicity and sensitization, does not cause skin photosensitive reaction, can obviously improve apoptosis and necrosis caused by ultraviolet irradiation, reduce inflammatory exudation of dermis and degradation of collagen and elastic fiber, reduce adverse reactions of erythema, inflammation, exudation, desquamation, scabbing and the like caused by large-dose ultraviolet irradiation of skin, and effectively relieve sunburn of the skin.

Comparative example 1

Preparing sargassum fusiforme dry powder: and (3) taking the dried sargassum fusiforme fronds, washing the sargassum fusiforme fronds for 3-4 times by using deionized water, and then drying and crushing the sargassum fusiforme fronds to obtain the finished product.

Normal-temperature water extraction: weighing 4g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 160mL of distilled water, uniformly mixing, and standing for 1h at normal temperature. Centrifuging at 3000g for 15min, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and lyophilizing the retentate to obtain Sargassum extract (RM water).

Leaching with boiling water: weighing 4g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 160mL of distilled water, uniformly mixing, and carrying out water bath at 100 ℃ for 1 h. Cooling the reaction solution to room temperature, centrifuging for 15min at 3000g, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and lyophilizing the cut-off solution to obtain seaweed extract (binding water).

Leaching with dilute alkali: weighing 4g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 160mL of 0.1mol/L NaOH solution, uniformly mixing, and carrying out water bath at 90 ℃ for 1 h. Cooling the reaction solution to room temperature, centrifuging at 3000g for 15min, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and lyophilizing the cut-off solution to obtain Sargassum extract (NaOH).

Leaching with dilute acid: weighing 4g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 160mL of 0.1mol/L HCl solution, uniformly mixing, and carrying out water bath at 90 ℃ for 1 h. Cooling the reaction solution to room temperature, centrifuging at 3000g for 15min, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and lyophilizing the cut-off solution to obtain Sargassum extract (HCl).

Ultrasonic treatment: weighing 4g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a beaker, adding 160mL of distilled water, and carrying out ultrasonic treatment for 1h (with the power of 120W) at 50 ℃. Centrifuging at 3000g for 15min, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and lyophilizing the retentate to obtain Sargassum extract (ultrasonic wave).

Enzymolysis of cellulase: weighing 2g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 80mL of 0.05mol/L, adjusting the pH value to 4 by using dilute hydrochloric acid, using cellulose protease (3000U/g seaweed) to carry out water bath at 37 ℃ for 1 h. Cooling the reaction solution to room temperature, centrifuging for 15min at 3000g, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and freeze-drying the cut-off solution to obtain the seaweed extract (cellulose).

And (3) enzymolysis by pectinase: weighing 2g of sargassum fusiforme dry powder, putting the sargassum fusiforme dry powder into a conical flask, adding 80mL of 0.05mol/L, adjusting the pH value to 3 by using dilute hydrochloric acid, using pectinase (3000U/g seaweed) to carry out water bath at 37 ℃ for 1 h. Cooling the reaction solution to room temperature, centrifuging at 3000g for 15min, filtering the supernatant, ultrafiltering (with molecular weight cutoff of 3kDa), and lyophilizing the retentate to obtain seaweed extract (pectase).

Carrying out enzymolysis by helicase: weighing 2g of Cyrtymenia Sparsa dry powder, placing into a conical flask, adding 80mL of 0.05mol/L, adjusting pH with dilute hydrochloric acid 6, snailase (3000U/g seaweed), and water bathing at 37 deg.C for 1 h. Cooling the reaction solution to room temperature, centrifuging for 15min at 3000g, filtering the supernatant, ultrafiltering (molecular weight cut-off of 3kDa), and freeze-drying the cut-off solution to obtain the seaweed extract (snailase).

The seaweed extracts prepared by the different methods are subjected to antioxidant activity evaluation:

(1) DPPH radical scavenging activity assay: placing 0.6mL of seaweed extract solution (200. mu.g/mL) into a 10mL test tube with a plug, adding 1.4mL of distilled water and 2mL of 0.1mol/L of DPPH ethanol solution, shaking, mixing uniformly, and carrying out water bath at 37 ℃ in a dark place for 30 min. After completion of the reaction, the absorbance (As) was measured at 517 nm. The mixture of 2mL of distilled water and 2mL of absolute ethyl alcohol is used for zero adjustment, and the blank control is 2mL of distilled water and 2mL of a 0.1mol/L ethanol solution of DPPH, and the absorbance at a wavelength of 517nm was designated as (Ac). Each sample was tested in parallel 3 times, and the inhibition ratio (%) of DPPH radical was (1-As/Ac). times.100%, As was A measured for the sample _517nm Ac is blank A _517nm 。

(2) Determination of superoxide anion radical scavenging activity: 0.4mL of the seaweed extract solution (200. mu.g/mL) was placed in a 10mL tube with a stoppered scale, and 0.6mL of Tris-HCl buffer (16mmol/L, pH8), 1mL of 78. mu. mol/L NADH solution (solvent 16mmol/L, pH8 Tris-HCl buffer), 1mL of 50. mu. mol/L NBT solution (solvent 16mmol/L, pH8 Tris-HCl buffer) and 1mL of 10. mu. mol/L PMS solution (solvent 16mmol/L, pH8 Tris-HCl buffer) were added. After shaking and mixing, the mixture is bathed in water at 25 ℃ for 5 min. After the reaction was completed, the absorbance (As) was measured at a wavelength of 560 nm. The blank was zeroed with Tris-HCl buffer (16mmol/L, pH8) and the same volume of the test solution was replaced with 0.4mL Tris-HCl buffer (16mmol/L, pH8) and the rest remained unchanged. Blank control the absorbance of the reaction mixture at 560nm was recorded as (Ac). Parallel 3 times per sample, for O ₂ ^- The inhibition ratio (%) of (1-As/Ac). times.100%, As being the result of measurement of A _560nm Ac is blank A _560nm 。

(3) Measurement of hydroxyl radical scavenging (OH.) Activity: placing 0.06mL seaweed extract solution (200 μ g/mL) in 10mL tube with plug, adding 0.04mL distilled water and 0.2mL KH ₂ PO ₄ KOH buffer (100mmol/L, pH7.4), 0.2mL of 15mmol/L alpha-deoxyribose, 0.2mL of 500mmol/L FeCl ₃ Solution, 0.1mL of 1mmol/L EDTA solution, 0.1mL of 1mmol/L vitamin C solution, 0.1mL of 10mmol/L H ₂ O ₂ The solution is shaken and mixed evenly and is bathed in water at 37 ℃ for 1 h. After the reaction, 1mL of 1% (w/v) TBA solution and 1mL of 2.8% (w/v) TCA solution were added to the mixture, mixed, and then subjected to 80 ℃ water bath for 20 min. The mixture was measured for absorbance (As) at a wavelength of 532 nm. The test solution was zeroed with distilled water, and the blank was replaced with 0.06mL of distilled water for the same volume of test solution, while the others remained unchanged. The absorbance of the blank reaction mixture at 532nm was recorded as (Ac). Each sample was subjected to parallel measurement 3 times, and the OH.degree (%) was (1-As/Ac). times.100%, and As was measured for each sampleA _532nm Ac is blank A _532nm 。

(4) Linoleic acid peroxidation inhibition assay: 1mL of the seaweed extract solution (200. mu.g/mL) was placed in a 10mL test tube with a stoppered mark, 4mL of PBS buffer (0.04mol/L, pH7.0) and 5mL of linoleic acid emulsion (containing 0.15% linoleic acid, 0.154% Tween-20, and the solvent being PBS buffer) were added, the mixture was shaken and mixed, and the mixture was washed with water at 37 ℃ for 12 hours. After the reaction is finished, 0.1mL of reaction mixture is placed in a 10mL test tube with a plug, 4.7mL of 75% (v/v) ethanol solution, 0.1mL of 30% (w/v) ammonium thiocyanate solution and 0.1mL of 0.02mol/L FeCl are added ₂ Solution (solvent 3.5% v/v hydrochloric acid solution). The mixture was rapidly mixed and the absorbance (As) was measured at a wavelength of 500 nm. The mixture of 0.3mL of distilled water and 4.7mL of 75% (v/v) ethanol solution was used for zeroing, and 1mL of PBS buffer (0.04mol/L, pH7.0) was used for the blank control instead of the same volume of the test solution, and the rest was kept unchanged. The absorbance of the blank reaction mixture at 500nm was recorded as (Ac). Each sample was tested in parallel 3 times, and the inhibition ratio (%) of linoleic acid peroxidation was (1-As/Ac). times.100%, As was A measured for the sample _500nm Ac is blank A _500nm 。

(5) Reduction of Fe ³⁺ And (3) capacity measurement: placing 0.06mL seaweed extract solution (200 μ g/mL) in 10mL tube with plug, adding 0.04mL distilled water, 3mL reaction solution (300 mL 300mmol/L, pH3.6 acetic acid buffer solution, 30mL 100mmol/L TPTZ solution, 30mL 20mmol/L FeCl ₃ Mixing the solution, and mixing the solution when the solution is prepared at present). After the mixture was subjected to water bath at 37 ℃ for 5min, the absorbance (As) was measured at a wavelength of 593 nm. The solution is adjusted to zero by using 300mmol/L acetate buffer solution with pH3.6, 0.06mL of distilled water is used for replacing the blank control with the solution to be detected with the same volume, and the rest is kept unchanged. The absorbance of the blank reaction mixture at 593nm was recorded as (Ac). Each sample was run in parallel 3 times for Fe ³⁺ Percent reduction (%) (1-As/Ac). times.100%, As is A measured for the sample _593nm Ac is blank A _593nm 。

(6) And (3) measuring the total antioxidant capacity: placing 0.03mL of seaweed extract solution (200. mu.g/mL) in a 10mL test tube with a plug, adding 3mL of reaction solution (180 mL each of 7.0mmol/L ABTS solution and 2.45mmol/L potassium persulfate solution), mixing uniformly for 12h in the dark, adding 100mmol/L,pH7.4 KH ₂ PO ₄ adjusting the absorbance to 0.7 with KOH buffer solution, and mixing with shaking. Standing for 30min, and measuring the absorbance (As) of the reaction mixture at 734nm wavelength. Using 100mmol/L, pH7.4 KH ₂ PO ₄ The KOH buffer was zeroed and the blank was replaced with 0.03mL of distilled water for the same volume of test solution, the others were kept unchanged. The absorbance of the blank reaction mixture at 734nm was recorded as (Ac). Each sample was tested in parallel 3 times, and the inhibition ratio (%) for ABTS was (1-As/Ac). times.100%, As was A measured for the sample _734nm Ac is blank A734 _nm 。

It was found (figure 9) that the activity of the seaweed extracts obtained by dilute acid digestion (HCl) and cellulase enzymatic hydrolysis (cellulose) was better (P <0.05) than the seaweed extract obtained by boiling water digestion (foaming water) in scavenging DPPH free radical activity.

Compared with the seaweed extract prepared by boiling water extraction (boil water), the seaweed extract prepared by normal temperature water extraction (RM water), dilute acid extraction (HCl), cellulase enzymolysis (cellulose), pectinase enzymolysis (pectase) and snailase enzymolysis (snailase) has better activity (P <0.01) in the aspect of scavenging superoxide anion free radical activity.

The seaweed extract obtained by dilute alkali leaching (NaOH) has better activity (P <0.01) in scavenging ABTS free radical activity than the seaweed extract obtained by boiling water leaching (bathing).

The activity of the seaweed extracts obtained by dilute alkali leaching (NaOH) and dilute acid leaching (HCl) was better (P <0.01) than that of the seaweed extract obtained by boiling water leaching (foaming water) in terms of reduced iron ion activity.

The seaweed extract obtained by dilute acid leaching (HCl) is more active (P <0.01) than the seaweed extract obtained by boiling water leaching (foaming water) in scavenging hydroxyl radical activity.

The seaweed extract obtained by dilute acid extraction (HCl) has better activity (P <0.05) than the seaweed extract obtained by boiling water extraction (foaming water) in inhibiting linoleic acid peroxidation activity.

However, it is noted that, in the above 8 preparation methods, the seaweed extract prepared by boiling water extraction was not optimal in vitro antioxidant activity, but was most excellent in the efficacy of increasing the activity of uv-irradiated cells (fig. 10, measured with reference to the method of example 5) and relieving sunburn of mouse skin caused by uv irradiation (all significantly different from the seaweed extract prepared in the comparative example).

Comparative example 2

Ethanol precipitation: weighing 200g of edible sargassum fusiforme sun-dried algae meeting the use standard of related marine products in China, soaking in deionized water for 30min, cleaning for 3-4 times, and soaking in deionized water for 30min again. Mincing Sargassum fusiforme, adding 5L deionized water, and boiling with 100 deg.C boiling water for 1 h. After the leaching liquor is cooled, centrifuging for 10min at 10000 r/min. And sequentially filtering the centrifugal supernatant through a suction filter (filtration qualitative filter paper) and an ultrafiltration membrane with the molecular weight cutoff of 5 kDa. Concentrating the ultrafiltration retentate to 200mL, adding 600mL precooled 4 deg.C anhydrous ethanol, standing at 4 deg.C for precipitation for 24h, centrifuging 3000g for 15min, washing the precipitate with anhydrous ethanol for 1-2 times, lyophilizing to obtain Sargassum extract YS75, concentrating the supernatant, and lyophilizing to obtain Sargassum extract YM.

The algal extracts YS75 and YM obtained in the present comparative example and the algal extract (YS) obtained in example 3 were subjected to cell activity evaluation using the experimental methods of examples 4 and 5.

It was found (FIG. 11) that at low doses (50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL), the seaweed extract (YM) prepared in the comparative example increased HaCaT cell activity more than the seaweed extract (YS) prepared in example 3; however, at high doses (800. mu.g/mL, 1600. mu.g/mL), the algal extract (YS) prepared in example 3 increased HaCaT cell activity significantly higher than the algal extracts YM, YS75 prepared in the comparative examples (P < 0.01).

Let us find it surprising (FIG. 12) that at the doses (50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 800. mu.g/mL, 1600. mu.g/mL) the seaweed extract (YS) obtained as prepared in example 3 increased the UVB-irradiated HaCaT cell activity significantly higher than the seaweed extracts YM, YS75 and the UVB model group (P <0.05 or P <0.01) obtained as prepared in the comparative example.

It can be seen that the effect of mitigating cell damage caused by uv irradiation was not significantly improved, but rather the efficacy of the two components (pellet, supernatant) of the seaweed extract prepared by ethanol precipitation treatment was reduced, as compared to the seaweed extract YS obtained in example 3.

Comparative example 3

Boiling water extraction (no ultrafiltration): weighing 200g of edible sargassum fusiforme sun-dried algae meeting the use standard of related marine products in China, soaking in distilled water for 30min, cleaning for 3-4 times, and soaking in distilled water for 30min again. Mincing Sargassum fusiforme, adding 5L distilled water, and decocting with 100 deg.C boiling water for 1 h. After cooling the leach liquor, centrifugation was carried out for 10min at 10000 g. And (4) carrying out suction filtration (filtration qualitative filter paper) on the centrifugal supernatant, concentrating and freeze-drying the filtrate to obtain the seaweed extract YC.

Seaweed extract YS was obtained by preparing according to the method of example 3.

And according to the method of the embodiment 3, the filtered solution is filtered by an ultrafiltration membrane with the molecular weight cutoff of 5kDa, the filtered solution of the 5kDa ultrafiltration membrane is collected, and the seaweed extract YD is obtained after concentration and freeze-drying.

The algal extracts YC and YD obtained in the present comparative example and the algal extract YS obtained in example 3 were subjected to cell activity evaluation by the experimental method of example 5.

It was found (FIG. 13) that at the dose (100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 800. mu.g/mL, 1600. mu.g/mL) the algal extract YS prepared in example 3 increased UVB-irradiated HaCaT cell activity, all significantly higher than the algal extract YD, algal extract YC and UVB model groups prepared in the comparative examples (P <0.05 or P < 0.01).

In conclusion, the seaweed extract (retentate component) obtained by ultrafiltration has the best effect of relieving cell damage caused by ultraviolet irradiation, and the sunscreen effect of the seaweed extract is remarkably superior to that of the seaweed extract prepared without ultrafiltration and the seaweed extract of the ultrafiltration permeate component.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. A preparation method of a seaweed extract capable of effectively relieving skin sunburn is characterized by weighing a certain amount of seaweed, adding water with the mass-volume ratio of 20-50 times g/ml, leaching with boiling water for 0.5-4 h, centrifuging 3000-10000 g of the leaching liquor for 10-15 min after cooling, sequentially performing suction filtration and ultrafiltration with a 3k-5kDa ultrafiltration membrane on the centrifuged supernatant, collecting ultrafiltration trapped fluid, concentrating and freeze-drying to obtain the seaweed extract; the seaweed is Hizikia fusiforme.

2. The preparation method of claim 1, wherein hizikia fusiforme is weighed, water with the mass volume ratio of 25 times g/ml is added, the mixture is extracted for 1 hour by boiling water, the extracted solution is cooled and then centrifuged for 10 minutes by 10000g, the centrifuged supernatant is sequentially filtered by suction and 5kDa ultrafiltration membranes, the ultrafiltration trapped fluid is collected, concentrated and freeze-dried, and the hizikia fusiforme is obtained.

3. The seaweed extract prepared by the method for preparing a seaweed extract according to claim 1 or 2.

4. Use of the seaweed extract of claim 3 for the preparation of a medicament, cosmetic or biomedical material for the prevention and/or treatment of sunburn of the skin.

5. The use of claim 4, wherein the medicament is in the form of a lotion, ointment, tincture, spirit, powder, oil, paste, plaster, film or aerosol; the seaweed extract accounts for 0.01-0.5% of the mass of the medicine.

6. The use of claim 4, wherein the cosmetic formulation comprises a cream, lotion, gel, lotion, or spray; the mass fraction of the seaweed extract in the cosmetic is 0.02% -0.5%.

7. The use of claim 4, wherein the dosage form of biomedical material comprises a dressing; the seaweed extract accounts for 0.02-0.5% of the mass of the biomedical material.

8. A pharmaceutical, cosmetic or biomedical material effective for relieving sunburn of the skin, characterized in that its active ingredient comprises the seaweed extract of claim 3.