阅读说明：本技术 苦茶碱在制备抗皮肤组织光损伤的产品中的应用 (Application of theophylline in preparation of product for resisting skin tissue photodamage ) 是由 何咏 李红建 胡云峰 吴实 刘赛君 郑忻凯 邓列华 于 2021-09-15 设计创作，主要内容包括：本发明公开了苦茶碱在制备抗皮肤组织光损伤的产品中的应用,所述皮肤组织光损伤是由紫外光线照射导致的。本发明发现苦茶碱能对抗紫外光线导致的皮肤组织光损伤,且浓度越高效果越明显。能够有效的避免紫外光线导致的皮肤组织光损伤,减少其造成的皮肤红斑、水肿,点状血管结构和毛细血管扩张,皮纹增粗,表面多发脱屑；有效干预角质层增厚,减少晒伤细胞产生,胞浆红染、核固缩、核溶、核碎、棘层细胞气球变性以及真皮层弥漫性炎症细胞浸润等皮肤细胞晒伤现象。较于其他天然化合物,苦茶碱取材容易,稳定性强,价格经济,且我国具有原产地优势和出口优势,有很好的经济前景,是值得深度研究开发的预防及治疗光损伤的原材料。(The invention discloses application of theophylline in preparing a product for resisting skin tissue photodamage caused by ultraviolet ray irradiation. The invention discovers that the theophylline can resist skin tissue light injury caused by ultraviolet rays, and the effect is more obvious when the concentration is higher. The ultraviolet light-induced skin tissue photodamage can be effectively avoided, and skin erythema, edema, punctate vascular structure and capillary vessel dilatation, dermatoglyph thickening and multiple desquamation on the surface caused by the ultraviolet light-induced skin tissue photodamage can be reduced; effectively intervenes in the thickening of the horny layer, reduces the skin cell sunburn phenomena such as the generation of sunburn cells, cytoplasmic red staining, nuclear shrinkage, nuclear lysis, nuclear fragmentation, acanthocyte balloon degeneration, dermal diffuse inflammatory cell infiltration and the like. Compared with other natural compounds, the theophylline has the advantages of easily obtained materials, strong stability and economic price, and China has the advantages of origin and export, has good economic prospect, and is a raw material for preventing and treating photodamage worthy of deep research and development.)

1. Use of theophylline for the manufacture of a product for the treatment of photodamage to skin tissue, wherein said photodamage to skin tissue is caused by irradiation with ultraviolet light.

2. Use according to claim 1, wherein the photodamage of skin tissue is apoptosis, inflammatory reaction, and/or oxidative stress.

3. Use according to claim 1, wherein the photodamage of skin tissue is skin swelling, purpura, and/or thickening.

4. The use according to claim 1, wherein the photodamage to skin tissue is telangiectasia of the skin, alterations in the vascular structure of the skin, erythema formation on the vascular lining of the skin, increased desquamation on the skin surface, decreased hair on the skin surface, increased thickness of the skin texture, and/or disorganization of the skin texture.

5. The use according to claim 1, wherein the photodamage to skin tissue is a marked thickening of the stratum corneum, increased sunburn cells, and/or increased inflammatory cells of the dermis.

6. The use according to claim 1, wherein the photodamage to skin tissue is one or more of cytoplasmic red staining, nuclear pyknosis, nuclear lysis, or nuclear fragmentation of skin keratinocytes.

7. The use of claim 1, wherein the photodamage to skin tissue is acanthocyte balloon degeneration.

8. The use according to claim 1, wherein the photodamage of skin tissue is diffuse inflammatory cell infiltration of the dermal layer.

9. The use according to claim 1, wherein the photodamage to skin tissue is an increase in the ratio of the expression level of the apoptosis-related protein Bcl-2 to the expression level of Bax.

10. The use of claim 1, wherein the photodamage to skin tissue is activation of the apoptosis-related protein caspase3 to clear-caspase-3.

Technical Field

The invention relates to the technical field of natural compound theophylline, in particular to application of theophylline in preparing a product for resisting skin tissue photodamage.

Background

Skin is the outermost organ of the human body and is susceptible to environmental damage upon repeated exposure to sunlight. Ultraviolet rays, which are the main components of sunlight-induced human skin damage, can pass through the epidermis to reach the epithelial layer of the dermis, causing DNA damage and increased oxidative stress, initiating cell signal transduction pathways, inducing expression of specific genes, and finally causing expression and activation of various intracellular protein kinases, various cytokines and matrix metalloproteinases, causing acute damage (sunburn ) and chronic cumulative damage (marine skin, actinic keratosis, photoaging, skin cancer) to the skin. Due to the destruction of the ozone layer, UVB radiation increases on earth, which exacerbates the risk of environmental damage to the skin and has long-term consequences such as photoaging, photo-immunosuppression, and photo-carcinogenesis. In addition, as the aging speed of the population is increased, the influence of skin photodamage on the skin health of the elderly is more obvious, and the incidence rate of light-related skin tumors is increased year by year.

Current products for combating ultraviolet light are primarily topical sunscreen products which protect the skin by absorbing or reflecting UV radiation from the surface of the skin. Sunscreens are divided into two broad classes based on their protective mechanisms: inorganic compounds and organic compounds. Inorganic compounds, which are inert particles capable of reflecting UVA and UVB radiation, mainly comprising zinc oxide and titanium dioxide, do not cause skin allergic reactions, but are visible on the skin surface, so inorganic sunblocks are undesirable from an aesthetic point of view. Organic sunscreens are generally carbonyl-conjugated aromatic compounds which absorb UV and release lower energy radiation, thereby protecting the skin from UV radiation. Organic sunscreens are not readily apparent on the skin surface and are more cosmetically acceptable, but many ingredients such as oxybenzone, sulindac are UV activated to produce photosensitive products which subsequently interact with the skin to cause adverse skin reactions. The incorporation of natural compounds into sunscreen formulations is therefore becoming increasingly important. In oxidative stress-mediated photodamage, the endogenous antioxidant capacity of the skin is a major determinant. Most natural compounds have antioxidation, and the natural compounds are safe to eat or externally used, and can prevent and reduce the occurrence and development of skin diseases caused by ultraviolet radiation.

Natural compounds are secondary metabolites produced by organisms found in nature. Topical application or consumption of natural compounds can prevent photodamage to the skin. Many natural organic substances can evolve upon exposure to intense radiation to form products with a variety of photoadaptation mechanisms, including the production of antioxidants and secondary metabolites that absorb ultraviolet light.

Wherein, the theophylline (1,3,7, 9-tetramethyluric acid, theocin) is a methylxanthine alkaloid, can be dissolved in chloroform and ethanol, is slightly soluble in water, and has good thermal stability. Mainly exists in bitter tea, and has certain sedative, hypnotic, anti-inflammatory and analgesic effects. CN101543498A discloses the application of theophylline in preparing anti-inflammatory analgesic drugs, specifically discloses that (1) the theophylline has certain function of resisting peripheral neuralgia: the high concentration of theophylline can produce analgesic effect 30min after administration, while the low concentration of theophylline has prolonged analgesic effect time, and may be related to its inhibition effect on central nervous system; (2) the theophylline has anti-inflammatory effect at high concentration.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides application of theophylline in preparing products for resisting skin tissue photodamage.

The invention aims to provide application of theophylline in preparing products for resisting skin tissue photodamage.

In order to achieve the purpose, the invention is realized by the following scheme:

the inventors found that UV can cause apoptosis of skin cells in a mouse model of photodamage to skin, and that theophylline can counteract this apoptosis and that the level of apoptosis in skin cells gradually decreases with increasing doses of theophylline. At the same time, the skin can be protected from damage caused by ultraviolet radiation by reducing inflammatory reaction, oxidative stress and influencing various signal paths.

The invention therefore claims the use of theophylline for the preparation of a product for combating photodamage to skin tissue, characterized in that said photodamage to skin tissue is caused by irradiation with ultraviolet light.

Preferably, the product is a pharmaceutical and/or nutraceutical product. The medicine and/or health care product is a protective agent for preventing and/or treating skin photodamage.

The product described herein contains, in addition to an effective amount of theophylline, the adjuvants necessary for the preparation of different dosage forms.

Preferably, the photodamage to skin tissue is apoptosis, inflammatory response, and/or oxidative stress. The theophylline can inhibit apoptosis, inflammatory response, and/or oxidative stress caused by photodamage to skin tissue.

Preferably, the photodamage to skin tissue is skin swelling, purpura, and/or thickening. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to skin tissue is telangiectasia of the skin, alterations in the vascular structure of the skin, formation of erythema on the skin due to flaking of blood vessels, increased desquamation on the skin surface, decreased hair on the skin surface, increased skin texture, and/or disorganized skin texture alignment. The theophylline can remarkably inhibit the above symptoms.

More preferably, the skin surface desquamation is a skin surface white fine particle desquamation. The theophylline can remarkably inhibit the above symptoms.

More preferably, the vascular structure is changed to a punctate, linear, and/or branched vascular structure change. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to skin tissue is a marked thickening of the stratum corneum, increased sunburn cells, and/or increased inflammatory cells of the dermis. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to the skin tissue is one or more of cytoplasmic red staining, nuclear pyknosis, nuclear lysis, or nuclear fragmentation of the skin keratinocytes. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to skin tissue is acanthocyte balloon degeneration. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to skin tissue is diffuse inflammatory cell infiltration of the dermal layer. The theophylline can remarkably inhibit the above symptoms.

Preferably, the skin tissue photodamage is increased ratio of apoptosis-related protein Bcl-2 to Bax expression. The theophylline can remarkably inhibit the above symptoms.

Preferably, the photodamage to skin tissue is activation of the apoptosis-related protein caspase3 to clear-caspase-3. The theophylline can remarkably inhibit the above symptoms.

The theophylline can obviously inhibit various symptoms of skin tissue photodamage, and has better inhibition effect than that along with the increase of dosage.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that the theophylline can resist skin tissue light injury caused by ultraviolet rays, and the effect is more obvious when the concentration is higher. The skin is effectively prevented from being erythematous and edematous due to the skin tissue light injury caused by ultraviolet rays, and the visible telangiectasis, punctate vascular structure and telangiectasia have more desquamation on the surface and thickened dermatoglyph; effectively intervenes in the obvious thickening of the cuticle, reduces the skin cell sunburn phenomena such as the generation of sunburn cells, cytoplasmic red staining, nucleus shrinkage, even karyolysis, nucleus fragmentation, acanthocyte balloon degeneration, diffuse inflammation cell infiltration of the dermis and the like. Compared with other natural compounds, the theophylline has the advantages of easily obtained materials, strong stability and economic price, and China has the advantages of origin and export, has good economic prospect, and is a raw material for preventing and treating photodamage worthy of deep research and development.

Drawings

FIG. 1 shows the general effect of theophylline in inhibiting UV damage to mouse skin.

FIG. 2 is a graph of the dermoscopic effect of theophylline on UV inhibition of photodamage to mouse skin (50X).

FIG. 3 is a graph of the pathological effect of theophylline on UV irradiation damaged mouse skin-HE staining (400X).

FIG. 4 shows the effect of theophylline on the expression of UV skin photodamage apoptosis-related proteins Bcl-2 and Bax; mean ± SD, n > 3P <0.05vs the Control; # P <0.05vs UV.

FIG. 5 is a graph of the effect of theophylline on UV skin photodamage apoptosis-related protein caspase3 expression; mean ± SD, n > 3P <0.05vs the Control; # P <0.05vs UV.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

(1) Experimental materials:

ICR mice, 6-8 weeks old, 25-30 g, SPF grade.

(2) Feeding experimental animals:

the experimental animals are ICR mice, and are raised in a 12h day/12 h night alternating common-grade animal raising room with the temperature of 24 +/-2 ℃ and the relative humidity of 50 +/-10%; the common granulated feed and clean tap water can be freely eaten; there are 10 per cage. Animals were housed in animal houses for 1 week of acclimatization prior to the official start of the trial.

(3) Preparing theophylline:

dissolving theophylline with pure water to obtain theophylline solution with concentration of 0.4g/L, 0.8g/L, and 1.6g/L respectively.

Example 1

First, experiment method

1. Experiment grouping

Taking 60 mice, randomly dividing into 6 groups, each group comprises 10 mice, and each female and male group comprises 5 mice, and respectively comprises blank control group (A group), UV control group (B group), theophylline control group (C group), theophylline low-dose group (D group), theophylline medium-dose group (E group) and theophylline high-dose group (F group).

The drug is administered according to groups before the UV light injury model is established. The theophylline group is administered with three different concentrations of theophylline solutions of 0.4g/L, 0.8g/L and 1.6g/L for 14 days (respectively as group D, group E and group F), the theophylline control group (group C) is administered with high concentration of theophylline solution for 14 days, and the blank control group (group A) and UV control group (group B) are administered with pure water for 14 days.

2. Establishing a UV light damage model:

carrying out abdominal anesthesia on experimental mice by using 10% chloral hydrate solution (4ml/kg), and depilating hair by using a depilatory cream after roughly shaving the back hairs of the mice by using a baby hair cutter; observing, evaluating and photographing; the UV lamp is turned on to stabilize the light source for 2 minutes, after the UV illuminometer confirms the irradiation intensity, the experimental mouse after gastric lavage is placed 30cm below the UV lamp, the back of the experimental mouse is upward, and the body position of the anesthetized experimental mouse is placed to fully expose the skin to avoid shadow shielding.

In dark environment, 1000mJ/cm of mice in UV control group (group B), low theophylline group (group D), medium theophylline group (group E) and high theophylline group (group F)²UV irradiation of/d, continuous irradiation for 14 days. The blank control group (group a) and the theophylline control group (group C) were not UV-irradiated.

3. Observation of mouse skin changes:

after 14 days of continuous daily gavage and UV irradiation, mice were subjected to a skin mirror examination of the bare skin on the back, a pathological biopsy (same site was taken) and euthanized, and recorded by photography.

4. The influence of the apoptosis protein is verified by a Western blot method:

samples are collected while the skin change of the mice is observed, and the influence of the expression of apoptosis-related proteins Bcl-2 and Bax and the expression of apoptosis-related proteins caspase-3 and cleared-caspase-3 of the mouse skin photodamage model after treatment of each group are detected by a Western blot immunoblotting method.

(1) Total protein extraction

The skin tissue was placed on ice, 100. mu.l of lysate was added, shaken well and placed on ice for 20min, during which time shaking was performed 2-3 times. After lysis, trituration was carried out and centrifugation was carried out at 14000rpm at 4 ℃ for 5 min. And (4) packaging the centrifuged supernatant into separate transfer-poured clean centrifuge tubes, and storing at-80 ℃.

(2) Protein concentration determination

Diluting the experimental sample: 2 μ L of each sample was added with 38 μ L H₂Dilution with O20-fold. Are arranged in sequence. Preparing a BCA reagent concentration determination working solution: taking 50 volumes of the solution A, adding 1 volume of the solution B, and fully and uniformly mixing to prepare the medicament for use. A96-well plate was prepared, and 20. mu.L each of the diluted standard sample and the experimental sample was placed in the 96-well plate. Add 200. mu.L of working solution to each well and incubate at 37 ℃ for 30min in the dark. Reading the light absorption value, namely the OD value in a microplate reader, wherein the wavelength is 560 nm. The protein solution from which the sample was extracted and 5 × loading buffer were mixed in a 4: 1 mixing, boiling for 5min, opening cover 1-2 times, and storing at-20 deg.C.

(3) SDS electrophoresis

Adding electrophoresis buffer solution into the electrophoresis tank to enable the electrophoresis solution inside the two pieces of glass to be higher than the sample loading hole, immersing the bottom of gel in the electrophoresis solution inside the electrophoresis tank at the outer side, enabling the liquid level inside the glass plate to be higher than the outer side, then loading the sample in sequence, ensuring that the total amount of protein in each hole is 20ug, and ensuring that the total sample loading amount is less than 30 uL. And (4) covering the cover of the electrophoresis tank, and after the anode and the cathode are subjected to constant-voltage electrophoresis at 80V until the bromophenol blue reaches the separation gel, performing constant-voltage electrophoresis at 120V until the bromophenol blue just comes out of the bottom of the separation gel. The electrophoresis time is about 2-3 h.

(4) Rotary film

After electrophoresis is finished, taking out gel, rinsing the gel in a membrane transferring buffer solution for several seconds, opening the electric transfer printing clamp in a sandwich mode, filling a special sponge soaked by the membrane transferring solution on each side, respectively placing an NC membrane soaked by the membrane transferring solution on cathode detection paper, removing bubbles, clamping the electric transfer printing clamp, filling the membrane transferring buffer solution into a transfer printing groove, inserting the transfer printing groove into a refrigerator, and ensuring that the NC membrane is close to the anode and the amino acid and the protein with the negative point move like the anode. At low temperature, 100V constant pressure membrane transfer is performed for 1 minute per 1kDa membrane transfer according to the protein molecular weight. The hybridization membrane was removed, rinsed for 5min with TBST, and blocked with 5% skim milk solution at room temperature for 1 hour. And washing the membrane for 10min by TBST.

(5) Immunological hybridization

First dressing and raising overnight at 4 ℃. Washing membrane for 10min × 4 times. The corresponding secondary antibody dilutions were incubated for 1h at 37 ℃. Washing membrane 10X 4 times. The hybridization membrane was placed on a transparent plastic plate, taking care not to allow the membrane to dry. The chemi-histochemical luminescent substrate was applied uniformly to the surface of the membrane using a clean pipette and the reaction was allowed to continue for 5 min. Excess substrate solution on the membrane surface was blotted off with filter paper supplied from the kit and placed in a cassette.

(6) Color development

ECL chemiluminescent liquid is adopted in the darkroom for color development, and all the following operations are carried out under a darkroom red light. The solutions A and B of ECL luminescence solution were prepared in a 1.5mL EP tube at a ratio of 1: 1. Taking out the X-ray film clip, cutting out the preservative film with proper size, and paving. The PVDF membrane was held up from the incubation box with forceps, and one corner of the membrane was first contacted with the filter paper, and excess TBST solution on the membrane was aspirated. The film is placed on a preservative film of an X-ray film clamp, the prepared ECL luminous liquid is dripped after the film is laid flat, and the preservative film is folded and covers the whole PVDF film. Taking out the X-ray film, cutting with scissors to obtain X-ray film with proper width, placing on the film covered by the fresh film, pressing down the X-ray film clip, and tabletting for 3-10 min. Taking out the X-ray film and putting the X-ray film into a developing machine quickly. After development was complete, the films were air dried at room temperature, scanned by a scanner, and the results were analyzed using Image J software.

Second, experimental results

1. Total effect of theophylline on inhibition of UV photodamage to mouse skin

The dorsal skin of each group of mice was observed at 2 time points before 14 days of continuous 1000mJ/d UV irradiation and after 14 days of irradiation, respectively. As shown in FIG. 1, the skin on the back of the placebo (group A) mice was normal and smooth. After the UV control group (group B) continuously irradiates for 14 days, the back of the mouse obviously swells, the color is ruddy and transparent, purpura can be seen locally, and the skin of the mouse can be obviously thickened when the mouse touches the middle. The skin on the back of the mice in the theophylline control group (group C) is normal and smooth, and is similar to the mice in the group A. The low theophylline group (group D) mice had a marked red and swollen skin on their backs,

2. skin mirror effect of theophylline for inhibiting UV (ultraviolet) light damage on mouse skin

The dorsal skin of each group of mice was subjected to a skin mirror observation after 14 days of continuous 1000mJ/d UV irradiation. As shown in fig. 2, the skin mirror of the back of the blank control group (group a) mice appeared pale red with a smooth surface. The appearance of the dermoscope of the theophylline control group (group C) is similar to that of the group A, and a normal skin image is shown. The UV control group (group B) had red skin, visible multi-directional telangiectasia, local visible punctate, linear and branched vascular structure change, blood vessels distributed in a sheet form to form erythema, white fine desquamation on the skin surface, local hair reduction, and skin texture thickening with disorder arrangement. The hypoderm of the low theophylline group (group D) shows erythema, visible punctate vascular structure, more desquamation on the surface and thickened skin lines. The bitter theophylline middle amount group (group E) has reduced erythema, light red color, thickened dermatoglyph, and white desquamation on surface compared with group D. The high-content group (group F) of theophylline has light red color under a skin mirror, slightly thickened dermatoglyph and no obvious desquamation on the surface.

3. Pathological Effect of Sophophylline on UV-irradiated damaged mouse skin

As shown in fig. 3, there was no obvious abnormality in the skin under the mirror of the mice in both the blank control group (group a) and the theophylline control group (group C); the UV control group (group B) and the low theophylline group (group D) showed significant thickening of the horny layer, more sunburn cells, red cytoplasmic staining, nuclear shrinkage, lysis, fragmentation, occasional balloon degeneration of cells in the spinous layer, increase of inflammatory cells in the dermis layer, and diffuse inflammatory cell infiltration in the dermis layer. The stratum corneum of the theophylline medium-volume group (group E) is thickened, and the increment is less than that of the group B and the group D, no obvious sunburn cells are seen, and inflammatory cells in the dermis are increased. No obvious abnormality was seen in the skin of the mice under the microscope in the high theophylline group (group F).

In conclusion, the theophylline can resist UV-induced skin tissue photodamage, and the effect is more obvious when the concentration is higher.

4. Effect of Theileline on apoptosis-related proteins

As shown in FIG. 4, Western blot immunoblotting was used to examine the effect of different doses of theophylline on the expression of apoptosis-related proteins Bcl-2 and Bax in a UV mouse skin photodamage model. Bcl-2 is an anti-apoptotic protein and Bax is a pro-apoptotic protein. As shown in the figure: with the increase of the dosage of the theophylline, the ratio of the anti-apoptotic protein Bcl-2 to the pro-apoptotic protein Bax gradually increases, and has statistical significance compared with the control group (P < 0.05). The results suggest that UV can cause apoptosis in skin cells in mouse models of photodamage to skin, whereas theophylline can counteract this apoptosis and the level of apoptosis in skin cells gradually decreases with increasing doses of theophylline.

As shown in FIG. 5, the expression of apoptosis-related proteins caspase-3 and clear-caspase-3 was detected after the skin damage of UV-induced mice by picophylline. During apoptosis, caspase3 may be activated into clear-caspase-3. The results show that: the bitter theophylline can obviously reduce the activation of caspase3, and the larger the bitter theophylline dosage is, the more obvious the inhibition activation effect is, and the results have statistical significance (P < 0.05). Therefore, the bitter theophylline can obviously reduce the apoptosis of skin cells caused by UV.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.