Use of deoxyvasicinone for the preparation of anti-aging preparations

Abstract

The application discloses application of deoxyvobtusine in preparation of anti-aging preparations and belongs to the technical field of biological medicines and cosmetic materials.The application creatively finds that deoxyvobtusine can be applied to the field of skin anti-aging, can promote proliferation of aged skin cells in vitro, reduces expression of aging-related beta-galactosidase, reduces the number of skin wrinkles of ICR mice in vivo, improves the epidermal thickness of aged skin, increases the content of collagen fibers, and has no obvious influence on main organs, which indicates that the deoxyvobtusine has good safety and in-vivo application potential.The Alkanna tinctoria is a traditional medicinal plant, and the deoxyvobtusine has good safety basis; meanwhile, the compound can also be obtained through microbial fermentation (such as marine Streptomyces), the source is sustainable, the extraction method is simple, the yield is stable, and the deoxyvobtusine is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical and cosmetic materials technology, specifically relating to the application of deoxydaunoside in the preparation of anti-aging agents. Background Technology

[0002] Skin aging is a complex biological process resulting from the combined effects of endogenous and exogenous factors. Its typical manifestations include skin laxity, decreased elasticity, wrinkle formation, pigmentation (such as melasma and age spots), and decreased skin barrier function. Excessive pigmentation is one of the most significant external signs of skin aging and a major skin concern for consumers. With the increasing trend of population aging and rising demands for healthy and aesthetically pleasing skin, the development of products that combine whitening, spot-fading, and anti-aging effects has become a hot topic in the cosmetics and pharmaceutical industries.

[0003] Currently, the main active ingredients in anti-aging and whitening products on the market include kojic acid, arbutin, niacinamide, and vitamin C derivatives. However, these ingredients often suffer from drawbacks such as poor stability, strong skin irritation, single target, or low transdermal absorption. For example, although kojic acid is an effective tyrosinase inhibitor, it is prone to oxidation and discoloration and may cause contact dermatitis. Therefore, developing novel, highly effective, and safe anti-aging active ingredients derived from natural plants has significant scientific and practical value.

[0004] Camel bonfire ( Peganum harmala *L.* (L.) belongs to the genus *L.* of the family Zygophyllaceae, mainly distributed in northwestern and northern my country. Its seeds are used in traditional medicine for dispelling wind and relieving pain, suppressing cough and asthma. *L.* seeds are rich in alkaloids, including β-carboline and quinazoline alkaloids. Deoxyvasicinone (also known as deoxyvasicinone) is one of its representative quinazoline alkaloids. Studies have found that deoxyvasicinone possesses anti-inflammatory, antidepressant, and antitumor activities. In recent years, this compound has also been isolated from marine-derived *Streptomyces*. However, the application of deoxyvasicinone in the field of anti-skin aging has not yet been reported. Summary of the Invention

[0005] To address the problems of poor stability, strong skin irritation, single target, or low transdermal absorption of existing anti-aging ingredients, this invention provides the application of deoxydaunoside in the preparation of anti-aging formulations. Deoxydaunoside is a natural source that can promote the proliferation of aging skin cells, reduce the expression of aging-related β-galactosidase, improve the thickness of the epidermis of aging skin, and increase collagen fiber content, and can be applied in the field of anti-skin aging.

[0006] This invention is achieved through the following technical solution: In a first aspect, this invention provides the application of deoxyvasicinone in the preparation of anti-aging agents, wherein the structural formula of deoxyvasicinone (also known as deoxyvasicinone) is: Its molecular formula is C 11 H 10 N2O, with a molecular weight of 186.21 and CAS number 530-53-0.

[0007] Furthermore, the deoxyduckbillone alkaloid was extracted from camel awning.

[0008] Furthermore, the extraction method of the deoxyduckbillone alkaloid includes the following steps: (1) The camel husk was dried and pulverized, and then extracted with an organic solvent to obtain a crude extract; (2) The crude extract was subjected to acid dissolution and alkali precipitation to obtain the total alkaloid fraction; (3) The total alkaloid fraction was subjected to column chromatography to obtain deoxyduckbillone alkaloid.

[0009] Further, the organic solvent in step (1) is 85% ethanol, and the extract is concentrated until there is no alcohol odor to obtain crude extract.

[0010] Further, the acid-dissolution and alkali-precipitation treatment method in step (2) is as follows: the crude extract is adjusted to pH 1-2 with 1% dilute hydrochloric acid, extracted three times with ethyl acetate (ethyl acetate: aqueous phase = 1:1), and concentrated under reduced pressure to obtain the aqueous phase and the ethyl acetate phase. The pH is adjusted to 9-10 with ammonia water, extracted six times with chloroform (chloroform: aqueous phase = 1:1), and concentrated under reduced pressure to obtain the chloroform phase.

[0011] Further, the column chromatography in step (3) consists of 100-200 mesh silica gel column chromatography, 300-400 mesh silica gel column chromatography and H mesh silica gel column chromatography, respectively.

[0012] Furthermore, the anti-aging preparation is an anti-aging drug or an anti-aging cosmetic.

[0013] Furthermore, the deoxydaunoside promotes the proliferation of senescent cells and reduces the expression of senescence-related β-galactosidase.

[0014] Furthermore, the deoxygenated duckbill ketone alkaloid reduces the number of skin wrinkles; improves the thickness of the epidermis of aging skin; and increases the content of collagen fibers.

[0015] Furthermore, the anti-aging preparation is applied to the skin surface.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: This invention creatively discovers that deoxyduckbillone alkaloids can be applied to the field of skin anti-aging. In vitro, it can promote the proliferation of aging skin cells and reduce the expression of aging-related β-galactosidase; in vivo, it reduces the number of wrinkles in ICR mice; improves the thickness of aging skin epidermis, and increases collagen fiber content, without significant effects on major organs, suggesting good safety and potential for in vivo application. Camelgrass is a traditional medicinal plant, therefore deoxyduckbillone alkaloids have a good safety profile; furthermore, this compound can also be obtained through microbial fermentation (such as with marine Streptomyces), making the source sustainable, the extraction method simple, and the yield stable, suitable for industrial production. Attached Figure Description

[0017] Figure 1 The mass spectrum of the deoxydaunoside prepared in Example 1; Figure 2 The deoxyduckbill ketone alkaloid prepared in Example 1 1 H-NMR spectrum; Figure 3 The deoxyduckbill ketone alkaloid prepared in Example 1 13 C-NMR spectrum; Figure 4 The graph shows the effect of different concentrations of deoxydaunodole ketone on the survival rate of HSF cells and HaCaT cells. A represents HSF cells and B represents HaCaT cells. Figure 5 Microscopic images and graphs showing the expression levels of β-galactosidase-related cellular senescence in different treatment groups are shown. A shows a microscopic image of β-galactosidase-related senescence in HSF cells of different treatment groups; B shows the expression levels of β-galactosidase in HSF cells of different treatment groups; C shows a microscopic image of β-galactosidase-related senescence in HaCaT cells of different treatment groups; and D shows the expression levels of β-galactosidase in HaCaT cells of different treatment groups. Figure 6 Images of the back skin of ICR mice after one week (W1), two weeks (W2), three weeks (W3), and four weeks (W4) of treatment; Figure 7 Image of H&E staining on the back skin of ICR mice; Figure 8 Image of Masson staining on the back skin of an ICR mouse. Detailed Implementation

[0018] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0020] Example 1 Extraction of deoxyvasicinone: (1) Take 30 kg of camel bonsai seeds, crush them, and soak them in 85% ethanol at room temperature for 4 times by the soaking method. Each cycle is 7 days. Collect the leachate, concentrate it under reduced pressure, and obtain 4 kg of total extract. (2) The total extract from step (1) was adjusted to pH 1.5 with 1% dilute hydrochloric acid, and extracted three times with ethyl acetate aqueous solution (ethyl acetate to water volume ratio of 1:1). The extract was concentrated under reduced pressure to obtain an aqueous phase and an ethyl acetate phase (1250 g). The pH of the ethyl acetate phase was adjusted to 9 with ammonia, and extracted six times with chloroform aqueous solution (chloroform to water volume ratio of 1:1). The extract was concentrated under reduced pressure to obtain a chloroform phase (1273 g). The chloroform phase sample was first preliminarily separated using D101 macroporous adsorption resin. After preliminary experiments, ethanol aqueous solutions with concentrations of 30%, 70%, and 90% were selected to elute the chloroform phase. After elution, the total chloroform phase sample was separated into three fractions: A, B, and C (corresponding to the above 30%, 70%, and 90% eluents, respectively). Fraction B (232.22 g) g) The fractions were separated by 100-200 mesh silica gel column chromatography with dichloromethane-methanol gradient elution (200:1→1:1, v / v). The fractions were combined by TLC thin-layer chromatography to obtain fractions B1 to B9. Fraction B1 (1973.5 mg) was separated by 300-400 mesh silica gel column chromatography with petroleum ether (PE)-ethyl acetate (EA) gradient elution (80:1→1:1, v / v) to obtain fractions B1-1 to B1-16. (3) In step (2), fraction B1-11 (812.1 mg) was separated by H-mesh silica gel column chromatography with gradient elution of petroleum ether (PE)-ethyl acetate (EA) (10:1→1:1, v / v) to obtain compound 1. t R = 8.8 min, 513.9 mg).

[0021] The mass spectrum of compound 1 obtained by the above preparation and separation is as follows: Figure 1 As shown, [M+H] + (m / z 187.0899); 1 H-NMR spectrum as shown Figure 2 As shown, 13 C-NMR spectrum as shown Figure 3 As shown, it was identified as deoxyduckbillone alkaloid, consistent with the data reported in the literature.

[0022] Example 2 In vitro anti-skin cell aging test of deoxyvasicinone (1) Human skin fibroblasts (HSF) and human epidermal keratinocytes (HaCaT) were seeded in 96-well plates and treated with different concentrations (0, 0.1, 1, and 10 μmol / L, final concentrations in the culture medium) of deoxyvasicinone for 6 h. Then, doxorubicin (DOX) was added (final concentrations after adding doxorubicinone were 0.2 μmol / L and 0.25 μmol / L, respectively) for 24 h to induce cell senescence. After that, the cells were cultured in normal culture medium for 24 h, and 10 μl CCK8 / 100 μl DMEM / well was added. The cells were incubated at 37℃ for 2 h. The absorbance at 450 nm was measured by microplate reader, and the cell viability was calculated. The effects of different concentrations of deoxyvasicinone on the viability of HSF and HaCaT cells were as follows: Figure 4 As shown (A represents HSF cells, B represents HaCaT cells), by Figure 4 It was found that Deoxyvasicinone treatment promoted the proliferation of HSF cells and HaCaT cells in a concentration-dependent manner, with the most significant effect at a concentration of 1 μmol / L.

[0023] (2) Human skin fibroblasts (HSF) and human epidermal keratinocytes (HaCaT) were seeded into 6-well plates and treated with doxorubicin (DOX) (final concentrations after adding doxorubicin were 0.2 μmol / L and 0.25 μmol / L, respectively) for 24 h to induce cell senescence. Then, the cells were cultured in normal medium for 24 h. The intracellular β-galactosidase content was detected using a cell senescence β-galactosidase staining kit. The specific detection method was as follows: wash once with PBS, add β-galactosidase staining fixative and fix at room temperature for 15 min, remove the fixative, wash the cells three times with PBS, add staining working solution, incubate overnight at 37 ℃, observe under a regular optical microscope, and take pictures for recording. At the same time, the lactase / β-galactosidase (β-GAL) activity detection kit was used: add 1 ml of reagent one for every 5 million cells, sonicate the cells (ice bath, power 200 W, sonication for 3 s, interval 10 s, repeated 30 times), and centrifuge at 12000 g and 4℃ for 10 minutes. After 30 minutes, the supernatant was collected and placed on ice for testing. Then, according to the kit instructions, reagents 1 and 2 were added to the sample, and the mixture was incubated at 37 °C for 30 minutes. Reagent 3 was then added, and the mixture was thoroughly mixed. The absorbance was measured at 400 nm. β-GAL activity for each group was determined according to the formula in the instructions and normalized. Microscopic images and β-galactosidase expression levels of aging-related β-galactosidase in different treatment groups (Control, aging model group (DOX), and Deoxyvasicinone treatment group (Deoxyvasicinone+DOX)) are shown below. Figure 5 As shown, A is a microscopic image of senescence-related β-galactosidase expression in HSF cells of different treatment groups, B is a graph of β-galactosidase expression levels in HSF cells of different treatment groups, C is a microscopic image of senescence-related β-galactosidase expression in HaCaT cells of different treatment groups, and D is a graph of β-galactosidase expression levels in HaCaT cells of different treatment groups. Figure 5 It can be seen that in both cell lines, the expression of senescent β-galactosidase was upregulated in the doxorubicin-induced senescence model group, while the addition of Deoxyvasicinone treatment could downregulate the expression of β-galactosidase in senescent cells.

[0024] The above results indicate that Deoxyvasicinone has an anti-skin cell aging effect in vitro.

[0025] Example 3 Tests of the anti-skin aging effects of deoxyvasicinone in ICR mice Male ICR mice aged 6-8 weeks were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd. After a week of acclimatization, subsequent experiments were conducted. The experiment included a control group (no treatment), a UVB group, a solvent + UVB group (20% propylene glycol, 200 μl propylene glycol + 800 μl purified water), and a Deoxyvasicinone + UVB group (10 mg / ml, 10 mg Deoxyvasicinone + 200 μl propylene glycol + 800 μl purified water), with 6 mice in each group. The UVB was applied half an hour before irradiation and every other day (2*2 cm of the back skin), 100 μL per mouse each time. The experiment lasted four weeks. The UVB irradiation dose in the first and second weeks was 540 mJ / cm². 2 The UVB radiation dose in the third and fourth weeks was 1080 mJ / cm². 2 Changes in the dorsal skin of mice were photographed at one week (W1), two weeks (W2), three weeks (W3), and four weeks (W4). Mice were sacrificed four weeks later, and dorsal skin was harvested. The skin tissue was fixed overnight in 4% histocellular fixative (Beyotime, Shanghai, China), then dehydrated, cleared, paraffin-embedded, and embedded before being cut into 5 μm sections. The sections were stained using a H&E staining kit and a modified Masson's trichrome staining kit, and photographed for observation. Images of the dorsal skin of ICR mice are shown below. Figure 6 As shown, by Figure 6 It was found that the skin on the backs of mice in the UVB group was rougher and drier than that in the control group, accompanied by skin laxity and increased wrinkles; the above conditions were significantly improved in the Deoxyvasicinone + UVB group; there was no significant difference between the solvent + UVB group and the UVB group.

[0026] H&E staining of the back skin yielded the following results: Figure 7 As shown, by Figure 7 It can be seen that the epidermis on the back of mice in the UVB group was significantly thicker than that in the control group (yellow arrow) and there was inflammatory cell infiltration (blue arrow). Compared with the UVB group, the epidermis in the Deoxyvasicinone + UVB group was thinner and the inflammatory cell infiltration was reduced. There was no significant difference between the solvent + UVB group and the UVB group.

[0027] Masson staining was performed on the skin on the back, and the results are as follows: Figure 8 As shown, by Figure 8 It can be seen that the UVB group had fewer collagen fibers than the control group, and there were breakage and irregular arrangement phenomena; the Deoxyvasicinone + UVB group had more collagen fibers and restored regular arrangement; the solvent + UVB group had no significant difference from the UVB group.

[0028] The above results indicate that deoxyvasicinone reduces the number of skin wrinkles in ICR mice, improves the thickness of the epidermis in aging skin, and increases the content of collagen fibers.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included within the protection scope of the present invention.

Claims

1. The application of deoxydaunoside in the preparation of anti-aging agents, characterized in that, The structural formula of the deoxy-duckbill ketone is: 。 2. The application of the deoxydaunoside alkaloid according to claim 1 in the preparation of anti-aging agents, characterized in that, The deoxyduckbillone alkaloid was extracted from camel awning.

3. The application of the deoxyduckbillone alkaloid according to claim 2 in the preparation of anti-aging agents, characterized in that, The extraction method of the deoxygenated duckbill ketone includes the following steps: (1) The camel husk was dried and pulverized, and then extracted with an organic solvent to obtain a crude extract; (2) The crude extract was subjected to acid dissolution and alkali precipitation to obtain the total alkaloid fraction; (3) The total alkaloid fraction was initially separated by macroporous adsorption resin and then subjected to silica gel column chromatography to obtain deoxyduckbill ketone.

4. The application of the deoxydaunoside alkaloid according to claim 3 in the preparation of anti-aging agents, characterized in that, The organic solvent in step (1) is 85% ethanol. The extract is concentrated until there is no alcohol odor to obtain crude extract.

5. The application of the deoxydaunoside according to claim 3 in the preparation of anti-aging agents, characterized in that, The acid-dissolution and alkali-precipitation treatment method in step (2) is as follows: the crude extract is adjusted to pH 1-2 with 1% dilute hydrochloric acid, extracted three times with ethyl acetate aqueous solution, concentrated under reduced pressure to obtain the aqueous phase and ethyl acetate phase; the pH is adjusted to 9-10 with ammonia water, extracted with chloroform aqueous solution, concentrated under reduced pressure to obtain the chloroform phase.

6. The application of the deoxyduckbillone alkaloid according to claim 3 in the preparation of anti-aging agents, characterized in that, The column chromatography in step (3) consists of 100-200 mesh silica gel column chromatography, 300-400 mesh silica gel column chromatography and H mesh silica gel column chromatography, respectively.

7. The application of the deoxydaunoside alkaloid according to claim 1 in the preparation of anti-aging agents, characterized in that, The anti-aging preparations mentioned are anti-aging drugs or anti-aging cosmetics.

8. The application of the deoxydaunoside alkaloid according to claim 1 in the preparation of anti-aging agents, characterized in that, The deoxyduckbillone alkaloid promotes the proliferation of senescent cells and reduces the expression of senescence-related β-galactosidase.

9. The application of the deoxyduckbillone alkaloid according to claim 1 in the preparation of anti-aging agents, characterized in that, The aforementioned deoxygenated duckbill ketone alkaloid reduces the number of skin wrinkles; improves the thickness of the epidermis in aging skin; and increases the content of collagen fibers.

10. The application of the deoxydaunoside alkaloid according to claim 1 in the preparation of anti-aging agents, characterized in that, The anti-aging preparation is applied to the skin surface.

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