A centella asiatica-derived carbon dot, a preparation method thereof, and application thereof in cosmetics

Abstract

The application belongs to the technical fields of nanobiomaterials and cosmetic efficacy materials, and discloses a centella asiatica-derived carbon dot, a preparation method thereof and application thereof in cosmetics. The preparation method of the centella asiatica-derived carbon dot comprises the following steps: step 1, using centella asiatica extract as a carbon source and urea as a nitrogen source, performing a hydrothermal carbonization reaction to obtain a reaction solution; and step 2, performing ultrafiltration purification and freeze-drying on the reaction solution obtained in step 1 to obtain the centella asiatica-derived carbon dot. The centella asiatica-derived carbon dot can penetrate the stratum corneum to achieve deep delivery to the dermis and form protection to the collagen matrix.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials and cosmetic efficacy ingredients, and in particular to a Centella asiatica carbon dot extract, its preparation method, and its application in cosmetics. This invention uses Centella asiatica extract as a precursor to prepare Centella asiatica-derived carbon dots (CeNDs) via a urea-assisted nitrogen-doped hydrothermal carbonization method. These carbon dots can be widely used in cosmetics with anti-glycation and collagen matrix protection functions. Background Technology

[0002] Skin glycation is one of the important biochemical mechanisms that accelerate skin aging. Reducing sugars in the body (glucose, fructose, etc.) can undergo non-enzymatic Maillard reactions with amino acid residues of collagen and other proteins, successively going through the Schiff base and Amadori product stages, and finally irreversibly generating advanced glycation end products (AGEs).

[0003] The accumulation of AGEs in the dermis can cause dual damage to the skin's extracellular matrix: First, AGEs form irreversible covalent cross-links between collagen molecules, leading to increased rigidity and decreased elasticity of collagen fibers, resulting in changes in appearance such as yellowing and dullness of the skin; Second, after AGEs bind to RAGE receptors on the cell surface, they can activate inflammatory pathways such as NF-κB, upregulate the expression of matrix metalloproteinases, and further accelerate the degradation of the collagen network.

[0004] Centella asiatica contains active ingredients such as asiaticoside, asiaticoside, and asiatic acid, and is a commonly used active ingredient in repair cosmetics. However, traditional Centella asiatica extracts have limitations: the active monomers have relatively large molecular weights, making it difficult to reach the dermis layer where collagen is located to exert a direct protective effect; at the same time, traditional extracts have insufficient ability to directly block AGEs.

[0005] Carbon dots (CDs) are a class of zero-dimensional carbon nanomaterials with particle sizes typically between 1 and 10 nm. They have advantages such as good water solubility, high biocompatibility, and strong antioxidant activity.

[0006] Based on the above background, this invention uses Centella asiatica extract as a carbon source and combines nitrogen-doped hydrothermal carbonization technology to prepare Centella asiatica-derived carbon dots (CeNDs), organically integrating the original efficacy of Centella asiatica with the characteristics of carbon dot nanomaterials to achieve multi-target intervention in the collagen glycation process. Summary of the Invention

[0007] The purpose of this invention is to address the problems of insufficient skin permeability and weak direct blocking ability of Centella asiatica extract in the prior art, and to provide a Centella asiatica-derived carbon dot, its preparation method and its application in cosmetics. The Centella asiatica-derived carbon dot has both anti-glycation function and nanoscale skin permeability.

[0008] The technical solution adopted to achieve the purpose of this invention is: A method for preparing carbon dots derived from Centella asiatica includes the following steps: Step 1: Using Centella asiatica extract as a carbon source and urea as a nitrogen source, a hydrothermal carbonization reaction is carried out to obtain a reaction solution; Step 2: The reaction solution obtained in Step 1 is purified by ultrafiltration and freeze-dried to obtain carbon dots from Centella asiatica.

[0009] In the above technical solution, in step 1, the total content of asiaticoside and hydroxyasiaticoside in the Centella asiatica extract is not less than 50% and the total content of asiatic acid and hydroxyasiatic acid is not less than 10% based on dry weight.

[0010] In the above technical solution, in step 1, Centella asiatica extract and urea are dissolved in ultrapure water at a mass ratio of 1:0.3 to 1:1 to prepare a homogeneous solution with a total concentration of 40 to 60 mg / mL, and then subjected to a hydrothermal carbonization reaction.

[0011] In the above technical solution, in step 1, the hydrothermal carbonization reaction is carried out in a sealed high-pressure reactor, the hydrothermal carbonization reaction temperature is 160~200℃, and the hydrothermal carbonization reaction time is 6~10 h.

[0012] In the above technical solution, step 2, the ultrafiltration purification step is as follows: the reaction solution is filtered sequentially through a 0.22 μm microporous membrane, centrifuged through a 3 kDa molecular weight cutoff ultrafiltration tube, and purified by dialysis through a 3500 Da molecular weight cutoff dialysis bag.

[0013] In the above technical solution, in step 2, the freeze-drying temperature is -50~-60℃, and the freeze-drying time is 15-35 h.

[0014] Another aspect of the present invention includes a carbon dot derived from Centella asiatica obtained using the preparation method described above.

[0015] Another aspect of the present invention includes the application of the Centella asiatica-derived carbon dots in cosmetics.

[0016] Another aspect of the present invention includes a cosmetic product whose active ingredient comprises the aforementioned Centella asiatica-derived carbon dots.

[0017] In the above technical solution, the amount of the Centella asiatica-derived carbon dots added to the cosmetic is 0.01 w / w%~2.0 w / w, and the pH range of the cosmetic is 5.0~7.0. During the preparation process, the Centella asiatica-derived carbon dots are added when the formulation temperature is below 45℃.

[0018] In the above technical solution, the functional components of the cosmetic also include Ganoderma lucidum-derived microvesicles and / or Cordyceps sinensis extract, constructing a synergistic skin care formula covering anti-glycation protection and collagen matrix reconstruction.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. Breakthrough in permeability: The carbon dots derived from Centella asiatica in this invention have a particle size of 2~10 nm, which can penetrate the stratum corneum to achieve deep delivery to the dermis. 2. Multi-target synergy: Covers multiple layers such as blocking AGEs formation and reducing intracellular AGEs formation, thus protecting the collagen matrix; 3. Compliance and Convenience: Using Centella asiatica extract as a precursor, the carbonized product can be declared in compliance with current cosmetic ingredient names, lowering the threshold for market entry and facilitating commercial promotion and application. Attached Figure Description

[0020] Figure 1 The transmission electron microscope image and particle size distribution histogram of the carbon dots (CeNDs) derived from Centella asiatica in this invention show that the carbon dots are spherical with a particle size concentrated in the range of 2 to 10 nm.

[0021] Figure 2 The hydrodynamic particle size stability monitoring (DLS) curves of CeNDs in PBS (pH 7.4) for 7 consecutive days demonstrate the good stability of the carbon dot colloids.

[0022] Figure 3 The results show the concentration-dependent inhibition of CeNDs on the fluorescence intensity (Ex 340 / Em430 nm) of AGEs in an in vitro collagen-glucose glycation model (day 14).

[0023] Figure 4 The results of qRT-PCR quantification of COL1A1, MMP-1 and NF-κB mRNA in high-glucose HaCaT cells after CeNDs treatment.

[0024] Figure 5 The graph shows the skin thickness (a) and hydroxyproline (HYP) content of each group in the mouse skin glycation model (b).

[0025] Figure 6Semi-quantitative statistical diagrams of immunohistochemical staining of AGEs, MMP-1 and COL1A1 in the skin of each group of the mouse skin glycation model (a) and MDA content (b) and SOD activity level in skin tissue homogenate (c).

[0026] Figure 7 Cytotoxicity evaluation of CeNDs (MTT assay, HaCaT cells, 0–200 μg / mL, 48 h). Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] Unless otherwise specified, all reagents used in the following examples are commercially available analytical grade products; all cells used are from the China Center for Type Culture Collection (CCTCC); and animal experiments are conducted in accordance with the regulations of the relevant animal ethics committees.

[0029] Example 1 A type of carbon dots (CeNDs) derived from Centella asiatica is prepared by the following steps: Step 1: Weigh 1.0 g of standardized Centella asiatica extract (centella asiatica glycoside + hydroxycentella asiatica glycoside not less than 50%, centella asiatic acid + hydroxycentella asiatic acid not less than 10%, dry weight) and 0.5 g of urea (as a nitrogen source to competitively bind with reducing sugars and protect collagen from glycation damage), add 20 mL of ultrapure water, stir magnetically until completely dissolved, and sonicate for 5 min to obtain a homogeneous and clear solution. Transfer the solution to a 50 mL polytetrafluoroethylene-lined autoclave, seal it, and place it in a 180℃ oven for 8 h. After naturally cooling to room temperature, obtain the reaction solution (brownish-yellow transparent liquid).

[0030] Step 2: The reaction solution obtained in Step 1 was filtered sequentially through a 0.22 μm microporous membrane, and then centrifuged and ultrafiltered using a 3 kDa molecular weight cutoff ultrafiltration tube (10000×g, 4℃, 30 min) to remove free small molecules. The retentate was collected and placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed in ultrapure water for 48 h (room temperature, water changed every 12 h) to remove inorganic ion impurities. The dialysate was freeze-dried (-50℃, 24 h) to obtain a pale yellow CeNDs powder, which was stored in a sealed container at 4℃ protected from light.

[0031] Comparative Example 1: Compared to Example 1, this comparative example did not add urea. 1.0 g of Centella asiatica extract of the same specification was weighed and dissolved in 20 mL of ultrapure water. Under the same conditions (180℃, 8 h), hydrothermal reaction, separation, and purification were carried out to obtain a nitrogen-free control sample.

[0032] Comparative Example 2 The raw material ratio (Centella asiatica extract 1.0 g + urea 0.5 g) was the same as in Example 1, but the hydrothermal reaction temperature was increased to 240℃ and the reaction time was 8 h. A deeply carbonized control sample was obtained through the same purification steps.

[0033] Example 2 Physicochemical characteristics of CeNDs: (1) Transmission electron microscopy (TEM) characterization: CeNDs were prepared into a 0.1 mg / mL aqueous solution, negatively stained with copper mesh, and observed under an accelerating voltage of 80 kV. CeNDs were spherical, with a particle size concentrated in the range of 2-8 nm and an average particle size of about 5.8 nm. Figure 1 ).

[0034] (2) Dynamic light scattering (DLS): The hydrated particle size of CeNDs (0.5 mg / mL, PBS, pH 7.4) was determined to be approximately 12 nm using a DLS particle size analyzer. Monitoring results over 7 consecutive days showed no significant change in particle size, indicating good colloidal stability. Figure 2 ).

[0035] (3) Zeta potential: CeNDs have a Zeta potential of approximately -22 mV (good electrostatic stability).

[0036] (4) Cytotoxicity (MTT assay): HaCaT cells were cultured for 24 h and then treated with 0–200 μg / mL CeNDs for 48 h. Results: The survival rate was 91.2% at 100 μg / mL and 83.6% at 200 μg / mL, with IC50 > 200 μg / mL, indicating good biocompatibility. Figure 7 ).

[0037] (5) Composition Analysis: In the preparation process of this invention, Centella asiatica extract and urea are added to the reaction at a mass ratio of 1:0.3 to 1:1 (preferably 1:0.5). Sufficient urea serves as a nitrogen-donating precursor, and under high-temperature and high-pressure hydrothermal pyrolysis, the resulting carbon dots have a high nitrogen doping content. These doped nitrogen elements are enriched on the surface of the carbon dots, manifesting as dense and abundant nitrogen-containing hydrophilic groups such as amino (-NH2) and amide groups. This significant and stable surface nitrogen content not only greatly improves the water solubility of the carbon dots but also provides sufficient "bait" active sites for competitive binding with reducing sugars and blocking saccharification reactions.

[0038] Example 3 In vitro anti-glycation experiment (collagen-glucose model): Prepare a collagen solution (type I collagen, 10 mg / mL, dissolved in 0.05 M phosphate buffer, pH 7.4), and add glucose to a final concentration of 0.5 M; (1) Add CeNDs prepared in Example 1 at different concentrations (0, 25, 50, 100, 200 μg / mL). When CeNDs is 0 μg / mL, it is recorded as the negative control group (collagen + glucose), and the rest are recorded as the model groups, namely CeNDs-25 μg / mL group, CeNDs-50 μg / mL group, CeNDs-100 μg / mL group, and CeNDs-200 μg / mL group; (2) Add aminoguanidine (AG, 1 mM) as a positive control group; aminoguanidine aqueous solution is strongly alkaline and decomposes upon heating to release ammonia gas, and is used as a positive control group for the experiment; (3) Add the control sample (100 μg / mL) obtained from Comparative Example 1 or Comparative Example 2. The experimental groups were sealed and incubated at 37°C in the dark for 14 days, with samples taken every 3 days. The characteristic fluorescence intensity of AGEs in each group (excitation 340 nm, emission 430 nm) was detected using a multi-functional microplate reader.

[0039] Results: After 14 days of incubation, the fluorescence intensity of AGEs in the model group was significantly higher than that in the negative control. The inhibition rates (100% - percentage of relative glycation degree) in the CeNDs treatment groups were approximately 27% for 25 μg / mL CeNDs, approximately 45% for 50 μg / mL CeNDs, approximately 55% for 100 μg / mL CeNDs, and approximately 63% for 200 μg / mL CeNDs, all reaching significant levels (p < 0.05), showing a concentration-dependent effect. The inhibition rate of the AG (1 mM) control was approximately 56%. CeNDs at 100 μg / mL was comparable to AG, and at 200 μg / mL it was significantly superior to AG. Figure 3 At a concentration of 100 μg / mL, Comparative Example 1 showed a fluorescence inhibition rate of only 15.4% against AGEs, failing to effectively block Schiff base formation due to the lack of "decoy" amino groups introduced by urea. Comparative Example 2 showed a fluorescence inhibition rate of only 21% against AGEs, also failing to effectively block Schiff base formation.

[0040] Example 4 CeNDs reduce the production of intracellular AGEs and their impact on gene expression: HaCaT cells were treated with 50 mM glucose for 24 h to establish a glycosylation model (designated as the model group) and cultured for another 24 h. The CeNDs-treated group was cultured in normal medium containing 100 μg / mL CeNDs for another 24 h. The blank control group (HaCaT cells) received no glycosylation treatment and were cultured in normal medium only. Intracellular AGEs levels showed that CeNDs significantly reduced AGEs production in HaCaT cells compared to the model group.

[0041] Gene expression: Total RNA was extracted from cells, reverse transcribed, and then subjected to qRT-PCR. Results: COL1A1 mRNA was upregulated by approximately 1.7-fold in the CeNDs-treated group compared to the model group, MMP-1 mRNA was decreased by approximately 41% (p < 0.05), and NF-κB mRNA was decreased by approximately 38% (p < 0.05). Figure 4 The difference in the reduction indicates that CeNDs not only directly inhibit the NF-κB pathway, but also generate a significant cascade amplification inhibition effect downstream through multi-target synergy, effectively blocking the enzymatic hydrolysis of the collagen matrix. The MMP-1 inhibition rate in Comparative Example 1 was only 16%, where inhibition rate = (model group expression level - comparative example group expression level) / model group expression level. The NF-κB inhibition rate was only 14% (41% and 38% respectively in Example 4), failing to exert a cascade amplification protective effect. Comparative Example 2, due to excessive high temperature, destroyed the natural pharmacological backbone that could coordinate with zinc ions; this sample had almost no direct inhibitory effect on MMP-1 and NF-κB (inhibition rate <10%).

[0042] Example 5 In vivo animal experiments: D-galactose-induced mouse skin glycation model.

[0043] Female C57BL / 6 mice were randomly divided into four groups: a blank control group, a model group (subcutaneous injection of D-galactose 100 mg / kg / day), a low-dose group (topical application of 0.05% CeNDs, 5 mg / kg), and a high-dose group (topical application of 0.10% CeNDs, 10 mg / kg). The CeNDs preparation was prepared using PBS solution containing 1% glycerol, while the other groups underwent topical treatment with PBS solution containing 1% glycerol in the same manner. Modeling lasted for 8 weeks, with the preparation applied topically to the dorsal skin daily starting from week 5.

[0044] Skin morphology and biochemical analysis: Skin thickness measurements showed that the skin thickness of mice in the high-dose group was significantly lower than that in the model group (p < 0.01); hydroxyproline (HYP) levels were significantly higher in the high-dose group than in the model group (p < 0.05). Figure 5 ).

[0045] Skin oxidative stress indicators: Skin tissue homogenate analysis showed that the MDA level in the high-dose CeNDs intervention group was reduced by approximately 42% compared to the model group (p < 0.05), and SOD activity was increased by approximately 1.8 times (p < 0.05), confirming the sustained antioxidant and matrix protective capabilities of CeNDs in living skin. Figure 6 ).

[0046] Immunohistochemistry (IHC): Compared with the model group, the high-dose CeNDs group showed a reduction of approximately 52% in the area of ​​skin AGEs positive, a reduction of approximately 49% in MMP-1 expression, and an upregulation of approximately 2.1-fold in COL1A1 expression (p < 0.01). Figure 6 The results showed that CeNDs effectively cleared the deposition of glycosylated products, inhibited the expression of matrix-degrading enzymes, and promoted collagen synthesis at the living tissue level.

[0047] Example 6 Examples of CeNDs cosmetic formulations Formula 1: CeNDs Anti-Glycation Serum (pH 6.0) Formulation composition (w / w): CeNDs (prepared in this invention) 0.10%, sodium hyaluronate 0.50%, nicotinamide 2.00%, glycerin 5.00%, 1,3-propanediol 3.00%, disodium EDTA 0.05%, phenoxyethanol 0.80%, carbomer 0.20%, triethanolamine (for pH adjustment) as needed, and deionized water as balance.

[0048] Preparation points: Carbomer is dispersed in deionized water, and other water-soluble components are added in sequence; the pH is adjusted to 5.5~6.0; the CeNDs stock solution is added after cooling to below 40℃, mixed well, filtered and filled.

[0049] Formula 2: CeNDs Repair Cream (pH 6.0) Formulation composition (w / w): CeNDs 0.20%, cetyl alcohol 2.00%, glyceryl stearate 3.00%, jojoba oil 5.00%, squalane 3.00%, glycerin 8.00%, 1,3-propanediol 4.00%, preservative 1.00%, carbomer 0.15%, triethanolamine as needed, deionized water balance.

[0050] Preparation points: The oil phase and aqueous phase are mixed and emulsified at 80°C; the CeNDs stock solution and preservative are added after cooling to below 45°C, and the pH is adjusted to 6.0. CeNDs must be added at a temperature below 45°C.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing carbon dots derived from Centella asiatica, characterized in that, Includes the following steps: Step 1: Using Centella asiatica extract as a carbon source and urea as a nitrogen source, a hydrothermal carbonization reaction is carried out to obtain a reaction solution; Step 2: The reaction solution obtained in Step 1 is purified by ultrafiltration and freeze-dried to obtain carbon dots from Centella asiatica.

2. The method for preparing carbon dots from Centella asiatica as described in claim 1, characterized in that, In step 1, Centella asiatica extract and urea are dissolved in ultrapure water at a mass ratio of 1:0.3 to 1:1 to prepare a homogeneous solution with a total concentration of 40 to 60 mg / mL, and then subjected to a hydrothermal carbonization reaction.

3. The method for preparing carbon dots derived from Centella asiatica as described in claim 1, characterized in that, In step 1, the hydrothermal carbonization reaction is carried out in a sealed high-pressure reactor at a temperature of 160-200°C for 6-10 hours.

4. The method for preparing carbon dots from Centella asiatica as described in claim 1, characterized in that, In step 2, the ultrafiltration purification process is as follows: the reaction solution is sequentially filtered through a 0.22 μm microporous membrane, centrifuged using a 3 kDa molecular weight cutoff ultrafiltration tube, and purified by dialysis using a 3500 Da molecular weight cutoff dialysis bag.

5. The method for preparing carbon dots derived from Centella asiatica as described in claim 1, characterized in that, In step 2, the freeze-drying temperature is -50~-60℃, and the freeze-drying time is 15-35 h.

6. Centella asiatica-derived carbon dots obtained by the preparation method according to any one of claims 1 to 5.

7. The application of the Centella asiatica-derived carbon dots as described in claim 6 in cosmetics.

8. A cosmetic product, characterized in that, Its active ingredients include the carbon dots derived from Centella asiatica as described in claim 6.

9. The cosmetic product as described in claim 8, characterized in that, The amount of the carbon dots derived from Centella asiatica added to the cosmetic is 0.01 w / w% to 2.0 w / w, and the pH range of the cosmetic is 5.0 to 7.

0.

10. The cosmetic product as described in claim 8, characterized in that, The active ingredients of the cosmetic also include Ganoderma lucidum-derived microvesicles and / or Cordyceps sinensis extract.

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