CN116211904B - Donggui extract and its extraction method and application - Google Patents

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to an Donggui extract, an extraction method and application thereof. The extraction method of the invention comprises the following steps: pulverizing radix Angelicae sinensis, adding into 70% ethanol solution, extracting under pressure, filtering under reduced pressure, centrifuging the filtrate, and collecting supernatant; performing ultrafiltration treatment on the supernatant by using a hollow cellulose acetate membrane, and obtaining ultrafiltrate after ultrafiltration is completed; concentrating the ultrafiltrate under reduced pressure to obtain concentrated solution of radix Angelicae sinensis, filtering the concentrated solution of radix Angelicae sinensis, subjecting the filtrate to chromatography with chromatography column containing polystyrene-divinylbenzene macroporous resin, eluting with water until the first eluent is neutral or sugar-free reaction is detected, eluting with eluent, and collecting the second eluent; concentrating the second eluent under reduced pressure to recover ethanol to obtain extract, and freeze-drying the extract. The invention also provides essence containing the Donggui extract, which has the effect of resisting skin aging.

Description

Donggui extract and its extraction method and application

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to an Donggui extract, an extraction method and application thereof.

Background

Aging of the skin and appearance is the most intuitive manifestation, and the national normal complexion should be reddish yellow, bright and moist, whereas facial skin aging is manifested as fine and dense wrinkles on the face, loose skin, dry desquamation, pigmentation, telangiectasia and hyperkeratosis. One report previously issued by market research company Zion Market Research shows that the global anti-aging skin care product market size reaches 2160 billion dollars in 2021, and is comparably increased by 7.5%. In China, however, it is generally accepted that China's anti-aging cosmetics consume over one hundred billion as a whole. Ai Mei data also shows that the internet search rate for anti-aging/anti-aging is as high as 300 hundred million times, and that 90% of chinese females have taken anti-aging measures, and that the anti-aging demands of chinese females are extremely strong. As the eastern Asian species and the caucasians are easier to color sink, the dermis is more compact and the face is flatter, and the skin has higher mechanical and chemical sensitivity compared with the caucasian species under the action of various factors such as irradiation, air temperature, living eating habit and the like. The research result of the skin aging of the Chinese shows that: the challenge of national anti-aging is not only wrinkles on the skin surface, but also the problem of more deeply focused contour collapse, and asians are more likely to get into the trouble due to flatter facial bones. The root of the problem is relieved, and the supporting force of the skin is improved, so that the definition of facial lines is improved, the skin state is tightened, and the visual aging weakness is relieved. Therefore, developing more efficient cosmetics against facial aging is a hot spot of concern to chinese scientists.

Dongda (A. Acutilloba (Sieb. Et Zucc) Kitagawa) is a perennial herb of the genus Angelica (Angelica) of the family Umbelliferae, which is also known as David and Gui, and is listed in Japan as the main variety of Angelica sinensis. As early as 70 s of 20 th century, eastern angelica was successfully introduced in yanjishi city of Jilin province in China. The chemical components of DongDang Gui mainly comprise ligustilide and its isomer, coumarin, sterol, polyacetylene, monosaccharide, polysaccharide, flavonoid and organic acid. Dong Dang Gui has the actions of strengthening spleen and keeping internal qi, replenishing blood and stopping bleeding, and activating blood and dredging collaterals, and is commonly used for treating menoxenia, amenorrhea, dysmenorrhea and other symptoms due to shaoyin blood deficiency and blood stasis.

Because of different extraction methods, the obtained Donggui extract has different active ingredients and effects. In the prior art, an extract of Donggui capable of effectively resisting skin aging is still lacking.

Disclosure of Invention

The first object of the present invention is to provide a method for extracting an extract of DongDang Gui, the second object of the present invention is to provide an extract of DongDang Gui extracted by the method, and the third object of the present invention is to provide an application of the extract of DongDang Gui.

According to a first aspect of the present invention, there is provided a method for extracting an extract of Donggui capable of effectively resisting skin aging, comprising the steps of:

(1) Pulverizing radix Angelicae sinensis, adding into 70% ethanol solution at a feed-liquid ratio of 1:10, pressurizing at 300MPa for 3min, vacuum filtering to obtain filtrate, centrifuging the filtrate, and collecting supernatant;

(2) Carrying out ultrafiltration treatment on the obtained supernatant, wherein a hollow cellulose acetate membrane is adopted as an ultrafiltration membrane, and ultrafiltrate is obtained after ultrafiltration is completed;

(3) Concentrating the obtained ultrafiltrate under reduced pressure to relative density of 1.15-1.25 (50deg.C), cooling to room temperature to obtain DongDang radix Angelicae sinensis concentrated solution, filtering to obtain filtrate, subjecting the filtrate to chromatography with chromatography column containing polystyrene-divinylbenzene macroporous resin, eluting with water until the first eluent is neutral or sugar-free reaction is detected, eluting with eluent, and collecting the second eluent; the eluent consists of the following components in percentage by weight: acetic acid 2%, ethanol water solution 98% with volume concentration of 75%; the composition of the eluent is determined according to the type of the active ingredient in the Donggui extract, thereby effectively eluting the target ingredient;

(4) Concentrating the second eluent under reduced pressure at 50deg.C to recover ethanol until the solution density at 25deg.C is 1.05-1.15 to obtain extract, and lyophilizing to obtain radix Angelicae sinensis extract.

In some embodiments, in step (1), the eastern angelica is pulverized into a fine powder having a particle size of 40 mesh.

In some embodiments, in step (1), the rotational speed of centrifugation is 5000r/min for 20min.

In some embodiments, in step (2), the hollow cellulose acetate membrane has a molecular weight of 50kD. The hollow cellulose acetate membrane of 45 kDa-55 kDa can effectively filter macromolecular impurities such as starch, and the invention adopts the hollow cellulose acetate membrane of 50kDa to carry out ultrafiltration treatment, thus being capable of obviously improving the clarity of the DongDang angelica extract and having a certain decoloring effect.

In some embodiments, in step (3), the Donggui concentrate is filtered with a 50 mesh screen.

In some embodiments, in step (3), the water elution flow rate is 2BV/h and the eluent elution flow rate is 2BV/h.

According to a second aspect of the present invention, there is provided an extract of DongDang Gui extracted by the above extraction method.

According to a third aspect of the present invention there is provided the use of an extract of Donggui as described above in the preparation of a cosmetic.

According to a fourth aspect of the present invention, there is provided an essence containing an extract of angelicae gigantis radix, comprising the following raw materials in parts by weight:

2.5% of the angelica sinensis extract, 5% of cyclohexyl glycerin, 3% of butanediol, 0.05% of hydrolyzed zinc hyaluronate, 0.05% of hydrolyzed calcium hyaluronate, 1% of N-acetylneuraminic acid, 0.05% of disodium edetate, 0.5% of p-hydroxyacetophenone, 0.2% of HY-1 acrylic acid polyresin, 0.4% of polyhexamethylene biguanide and the balance of water.

According to a fifth aspect of the present invention, there is provided a method for preparing the essence containing the extract of angelicae gigantis radix, comprising the steps of:

soaking zinc hyaluronate and calcium hyaluronate in water, fully swelling for 12h, adding water, dispersing uniformly, sequentially adding other components except polyhexamethylene biguanide and p-hydroxyacetophenone, stirring at a speed of 500r/min for 30min at 85 ℃, cooling to 40 ℃, adding polyhexamethylene biguanide and p-hydroxyacetophenone, and continuously stirring at a speed of 500r/min for 15 min.

Because the zinc hyaluronate and the calcium hyaluronate can not be fully dissolved in a short time, the zinc hyaluronate and the calcium hyaluronate can be fully dissolved by adopting a method of soaking and swelling by adding water in advance.

The beneficial effects of the invention include:

(1) According to the invention, the network medical framework and network pharmacology are utilized to predict the core components and key targets in the DongDang Gui, the molecular change of the drug intervention organism is subjected to multidimensional depiction, and further the analysis of complex drug effect components and drug effect mechanisms of the DongDang traditional Chinese medicine is realized systematically, so that the extraction method of the DongDang extract is determined according to the screened DongDang core components and key targets. The extraction method of the invention can obtain the skin aging resistant component in the angelica sinensis pertinently.

(2) According to the invention, the obtained DongDang extract is used for preparing the essence, and clinical test results show that the essence containing the DongDang extract can obviously improve the glossiness and the skin elasticity of the skin of a subject. From the subjective evaluation results of the subjects, the subjects showed that the product had various degrees of improvement on skin dryness, glossiness, dark yellow, firmness, elasticity and fine wrinkles after using the essence containing the extract of Donggui. Therefore, the essence containing the DongDang extract has the effect of resisting facial skin aging.

Drawings

Fig. 1 is a graph of similarity and aggregation characteristics of Donggui.

FIG. 2 is a feature panel diagram of LCC exploration of DongDang component target spots in the area of skin aging related proteins adjacent to the interaction group.

FIG. 3 is a graph showing the molecular effect and predicted therapeutic effect of the target of DongDang components on the basis of proximity and skin aging.

FIG. 4 is a functional group diagram of the search for key compounds of DongDang Gui by GEO analysis and cMAP ligation analysis.

FIG. 5 is a graph showing the expression level of the core gene of DongDang and the ROC curve thereof.

FIG. 6 is a diagram of the IncRNA-miRNA-target gene network of DongDang core gene and the therapeutic mechanism for treating skin aging.

FIG. 7 is a diagram showing the mechanism of action of the DongDang core component in resisting skin aging.

FIG. 8 shows the mass spectra of three controls of diosmin, zanthoxylum bungeanum toxin, and protocatechuic acid.

Fig. 9 is a total ion extraction flow chart of the extracts of the eastern angelica sinensis extracted in example 1 and comparative example 1, wherein fig. 9 (A, C) is a total ion extraction flow chart of the extracts of the eastern angelica sinensis extracted in example 1, and fig. 9 (B, D) is a total ion extraction flow chart of the extracts of the eastern angelica sinensis extracted in comparative example 1.

Fig. 10 is a graph comparing pre-treatment (left) and 28 days post-treatment (right) of subjects in a treatment group in a clinical trial of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but embodiments of the present invention are not limited thereto. The experimental materials and reagents referred to in the following examples are all available from commercial sources. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

According to the invention, the network medical framework and network pharmacology are utilized to predict the core components and key targets in the DongDang Gui, the molecular change of the drug intervention organism is subjected to multidimensional depiction, and further the analysis of complex drug effect components and drug effect mechanisms of the DongDang traditional Chinese medicine is realized systematically, so that the extraction method of the DongDang extract is determined according to the screened DongDang core components and key targets.

1. Screening of skin aging action mechanism and active ingredients of DongDang through lncRNA-miRNA-target gene network regulation based on network proximity exploration

1. Distribution characteristics and functional characteristics of Donggui component-target point

To explore the aggregation of target sites of the components of Dongyi in specific functional neighbors of the interaction group, by targeting targets of 29 components to the human interaction group, including 25121 proteins and 751939 interactions, 26 out of 29 Dongyi components were found to have two or more protein targets, while a few had abnormally high targets. Then, jaccard Index (JI) of each compound on protein target was calculated, and only limited similarity was found between targets of different DongDang Gui components (average JI=0.0115). The number of Donggui component-target binding and JI value distribution are shown in FIGS. 1A-C. This finding suggests that each component is directed to a specific set of proteins whose targets are limited to a common set of proteins, determined by the commonality of component binding domains in the three-dimensional structure of the protein target. The genomics enrichment analysis (fig. 1D) shows that the eastern angelica compounds regulate a common regulatory process, but their protein targets are less similar (average JI is lower), suggesting that they act against different pathways in the same biological process.

2. Dongganqi component-target specific area

To investigate whether the target of Dongyi is concentrated in a specific region of the human interaction group, dongyi components having more than two targets were studied with emphasis, and the size and significance of the Largest Connectivity Component (LCC) formed by each Dongyi component target was measured. It can be seen from fig. 2A that the target proteins of eastern angelica are aggregated in the vicinity of the skin aging-related proteins in the human protein interaction group. Further calculating LCC value of Dongyi ingredient target-skin aging target, wherein FIG. 2B is a bar graph of LCC value of Dongyi ingredient target in human skin aging protein interaction network, and FIG. 2C is size and significance of LCC formed by Dongyi ingredient target in the network, expressed as z value. The results showed that 15 LCCs out of 29 Dongyi components had happened to be larger than expected (z fraction > 1.95), the target tissues of these Dongyi components were among multiple size >2 connected components. These results indicate that the target of the Dongbing component modulates the well-positioned neighborhood specific to the interaction group. A network-based framework is thus sought to reveal the molecular mechanisms of specific eastern angelica components regulating health by exploring whether the regions of the interaction group targeted by eastern angelica are located within the network neighborhood associated with a particular disease.

3. The proximity between the target point of the component of the DongDang and the skin aging disease protein reveals that the DongDang has the therapeutic effect on skin aging

The Donggui components are regarded as drugs because they bind to specific proteins and have the ability to affect their normal functions. The research uses a network-based framework to predict the curative effect of the medicine on specific diseases, and can predict the curative effect of the Donggang component. The closer the target of the Dongyi ingredient is to the skin aging disease protein, the more likely the Dongyi ingredient will affect its phenotype. Thus, by calculating the network proximity between the Dongji component targets and the proteins associated with skin aging disorders, the nearest metric dc is used to represent the average shortest path length between each component target and the nearest skin aging related protein.

By calculating the distance, i.e., the proximity, between the network-based Donggui component target and 414 proteins associated with skin aging, the two groups of proteins were found to be very close to each other, and then the proximity between the component target and 1424 skin disease-related proteins was calculated. Ranking according to the network proximity of the target points of the components of the east Asian tree fern, and determining the components of the east Asian fern of 3 in the ranking: zanthoxylum toxin (Methoxsalen), protocatechuic acid (Protocatechuic acid), diosmin (Diosmin). The network proximity of the eastern angelica component was tested for skin aging disease and for differences in disease by comparing the toxicological genomics database (CTD) for 78 diseases for which treatment effects are known, by recalculating the proximity of the top 5 eastern angelica component to the 78 diseases validated, by evaluating the area under the curve (AUC) of the receive operating characteristics.

The PPI network diagram of the top three DongDang core component targets is shown in FIG. 3A, and the close interaction between the target proteins is seen, and the three components have non-negligible effects on skin diseases including skin aging and the like according to the proximity result (FIG. 3B). The results in fig. 3C show that the network proximity of the eastern angelica component is similar in skin aging disease and the validated disease AUC (auc=0.837), and that p=0.49 > 0.05, with no significant difference between the two groups, indicate that skin aging appears similar to the validated disease in the subject's working characteristic curve (ROC curve) analysis, and therefore it is believed that the eastern angelica active component also acts on skin aging, similar to the validated disease. Therefore, the first three Donggui components with the best prediction capability of the network medical framework on the treatment effect are primarily screened, the prediction performance is limited to the items with AUC of more than 0.6 and the prediction performance precision of more than 0.6.

4. Medicinal effect of DongDang core component for treating skin aging

Network proximity screening finds that three core components of the dioscin, the xanthotoxin and the protocatechuic acid in the angelica sinensis have the effect of targeted treatment on skin aging, and in order to further identify the specific effect and efficacy of the core components, the effect of the compounds is analyzed by gene disturbance by using a Connectivity Map database (cMAP, http:// www.broad.mit.edu/cMap /). First, 356 differential genes (adjust P <0.05, |logfc| > 0.5) were identified from the GSE192564 dataset in the GEO database, including 144 up-regulated genes and 212 down-regulated genes (fig. 4A-B). Venn analysis showed that the Donggui core component shares 29 intersection genes with disease differential genes (FIG. 4C), including 13 up-regulated genes and 16 down-regulated genes (FIG. 4D). cMAP database screening showed that the 3 AKK core components function similarly to BRD4770 inhibitor, doxorubicin, ML228, TW-37 inhibitor, BMY-45778, SRT1720, BRD-K30064966, arylretinoic acid, 1-phenyl biguanide and alfacalcidol (the first 10 components were selected according to ligation score, FIG. 4E). These components mainly have the functions of regulating aging epigenetic, resisting oxidation, regulating cell proliferation and apoptosis and regulating immunity, and all the mechanisms play important roles in inhibiting the progress of skin aging. Further explored the category of gene perturbation with the AKK core component connecting score greater than 60 points, the results show that the AKK core component is mainly related to BCL inhibitor, ATP synthase inhibitor, EGFR inhibitor, HIF activator, PKC activator, ribonucleotide reductase inhibitor, tubulin inhibitor, t-type calcium channel blocker, EIF protein LOF and PKC inhibitor (fig. 4F-G), suggesting that three core components of geraniin, xanthotoxin, protocatechuic acid in eastern angelica may be related to regulating metastasis, growth and apoptosis of cells, inhibiting oxidative stress, regulating energy metabolism of mitochondrial function, regulating vasodilation, and the like.

5. The occurrence and development of skin aging are related to the core target of AAK key compounds

Although 29 core genes have been previously obtained, it has not been determined whether these genes are actually involved in the disease process of skin aging. By protein-protein interaction (PPI) analysis, interactions were found between 29 core genes, focusing on key core targets for narrowing down, the first 12 key genes were selected according to the degree value of PPI (fig. 5C). Then, it was confirmed that in the test set (n=52), the expression of the proteins ABCC4, PTGER3, BCHE, HPGD, MOXD1 was up-regulated in the normal group, while the expression of AR, CXCR2, HSD17B2, ODC1, PI3, PLAU, THBS2 was down-regulated, and that the difference between the two groups was statistically significant (fig. 5A-B). This suggests that AKK key compounds may exert therapeutic effects by inhibiting or promoting the expression of these genes. Furthermore, ROC curves confirm that these core genes have a certain sensitivity and specificity for diagnosis of skin aging (AUC greater than 0.5, fig. 5D-G), especially AR, BCEH, CXCR, HPGD and PI3 perform well, with AUC greater than 0.9, suggesting that they can be specific biomarkers for diagnosis of skin aging. By calculating the Cutoff values, it was found that when the AR expression level was adjusted to 8.221, the BCHE expression level was adjusted to 9.948, the CXCR2 expression level was adjusted to 7.695, the HPGD expression level was adjusted to 9.686, and the PI3 expression level was adjusted to 7.603, the results were used as diagnostic criteria for skin aging.

6. lncRNA-miRNA-target gene regulation network of key target point of DongDang Gui core component

In eukaryotes MicroRNA (miRNA) is a class of small non-coding RNAs, approximately 22 nucleotides, targeting the 3 '-non-transcribed region (3' -UTR) of messenger RNA (mRNA) by base-complementary pairing (complete or incomplete pairing), directly degrading the messenger RNA or inhibiting translation, thereby completing post-transcriptional regulation of the target gene, and thus altering the genetic phenotype. Most long non-coding RNAs (lncrnas) have been identified as key regulatory factors for transcription and translation, playing a vital role in the normal functioning of cells.

The regulatory mechanism of the Donggui core target is discussed, as shown in fig. 6A, 19 regulatory core targets (such as AR, BCEH, CXCR, HPGD and PI 3) are screened from 6 miRNA databases, and mainly participate in cellular nitrogen compound metabolic process, small molecule metabolic process, cellular protein metabolic process, biosynthesis process, stress reaction, cellular protein modification process and immune system process, which are closely related to the progress of skin aging (fig. 6C). FIG. 6B shows that 90 lncRNAs (|energy| >25kCal/Mol, score > 150) including T086546, T018758, T179761, T088459, T088460, T088461, etc. are involved in the regulatory process of miRNAs on core targets. A transcription factor refers to a protein that binds to a specific nucleotide sequence upstream of a specific gene, which, together with miRNA, regulates the transcription of the gene by modulating RNA polymerase and DNA templates. By performing Transcription Factor (TF) enrichment analysis on miRNAs (fig. 6d, transmir v2.0 database), it was found that transcription factors ETS1, NFE2, FOXO1, KLF11, PBX1, ATF3, BRD3 and RFX5 are mainly involved in the regulation process of these 19 miRNAs, while AR is a transcription gene both TF and attached to the skin aging disease process. The results show that the core target genes of three core components of the geraniin, the xanthotoxin and the protocatechuic acid in the DongDang are displayed in the biological processes of the cellular nitrogen compound metabolic process, the small molecule metabolic process, the cellular protein metabolic process, the biological synthesis process, the stress reaction, the cellular protein modification process, the immune system process and the like through the lncrna-mirna-target gene network, and are one of the mechanisms for playing the role of treating skin aging diseases.

7. Mechanism of action and biological function of DongDang Gui core component

In order to further explore whether the action mechanism and biological function of three core components of the dioscin, the xanthotoxin and the protocatechuic acid in the east angelica are inhibited with the action of a core target point, single gene GSEA analysis is carried out on three core compounds of the dioscin, the xanthotoxin and the protocatechuic acid in the east angelica. The results indicate that both AR, BCEH, HPGD and PI3 are associated with up-regulation of autophagy and down-regulation of the phospholipase D signaling pathway, except CXCR2 (fig. 7). The results show that the three core components of the dioscin, the xanthotoxin and the protocatechuic acid in the angelica sinensis play the role of resisting skin aging through the mechanisms of the three core components.

2. Extraction of Donggui extract

Through the network medical framework based on the former part, the core components of the DongDang, which affect the skin aging process, are identified, and the mechanisms of the DongDang, such as the target points and the paths of the DongDang, which affect the skin aging process, are explored, and the extraction method of the DongDang is determined according to the three core components, namely the geraniin, the xanthotoxin and the protocatechuic acid.

Example 1

The extraction method of the DongDang extract of the embodiment comprises the following steps:

(1) The method comprises the steps of coarsely crushing DongDang radix Angelicae sinensis decoction pieces (purchased from Hebei Baicaokangshen medical company, guangzhong mountain university, a professor Wang Yonggang of modern Chinese medicine quality research development center) into DongDang radix Angelicae sinensis (root of A. Acutisloba (Sieb. Et Zucc) Kitagawa) belonging to Umbelliferae, taking 3kg of DongDang radix Angelicae sinensis powder, crushing into fine powder with granularity of 40 meshes, adding the fine powder into 70% ethanol solution according to a feed-liquid ratio of 1:10, filling into a vacuum packaging bag, and sealing to obtain ethanol solution of DongDang radix Angelicae sinensis powder.

(2) Pressurizing with small commercial ultrahigh pressure equipment (Tianjin Huataisen vast ultrahigh pressure equipment engineering Co., ltd.L 1-400/10) under 300MPa, extracting ethanol solution of Donggui powder for 3min at 25deg.C, pressure-relieving, filtering to obtain filtrate, centrifuging the filtrate at 5000r/min for 20min, and collecting supernatant.

(3) The obtained supernatant is subjected to ultrafiltration treatment by using a hollow fiber ultrafiltration membrane component (HFM-UF-90W external pressure type of He-Xin-Da membrane technology Co., ltd.) and the ultrafiltration membrane adopts a hollow cellulose acetate membrane with a molecular weight of 50kD, macromolecular impurities such as starch are filtered, and ultrafiltrate is obtained after ultrafiltration is completed. Therefore, the clarity of the DongDang extract can be obviously improved, and the DongDang extract has a certain decoloring effect.

(4) Concentrating the obtained ultrafiltrate under reduced pressure to a relative density of about 1.20 (50 ℃), cooling to room temperature to obtain an DongDang Gui concentrated solution, filtering the DongDang concentrated solution by a 50-mesh filter screen to obtain a filtrate, allowing the filtrate to pass through a chromatographic column filled with polystyrene-divinylbenzene macroporous resin to selectively adsorb active components in the filtrate, eluting the chromatographic column adsorbing the filtrate by 40BV water at a flow rate of 2BV/h until the first eluent is neutral, eluting by 60BV eluent at a flow rate of 2BV/h, wherein the eluent comprises the following components in percentage by weight: acetic acid 2%, ethanol water solution 98% with volume concentration of 75%, and collecting second eluent flowing out of the chromatographic column, wherein the second eluent is light yellow liquid.

(5) Concentrating the second eluent under reduced pressure at 50deg.C to recover ethanol until the solution density at 25deg.C is about 1.1 to obtain extract, and lyophilizing to obtain radix Angelicae sinensis extract with yield of 0.3wt%.

Comparative example 1

The extraction method of the Donggui extract of this comparative example is basically the same as that of example 1, except that in step (4), the filtrate is passed through a chromatography column packed with methacrylate macroporous resin.

Next, the components of the extract of Donggui obtained in example 1 were identified and the contents were measured.

The Donggui extract obtained in example 1 was prepared into a 10mg/mL sample solution using methanol as a solvent, and standard geranylgeranioside (purchased from Shanghai Seikovia Biotechnology Co., ltd., lot number: Y18N7C24715, purity greater than 95%), zanthoxylum bungeanum toxin (purchased from Shanghai Seikovia Biotechnology Co., ltd., lot number: P30J7M16998, purity greater than 98%), protocatechuic acid (purchased from Shanghai Seikovia Biotechnology Co., ltd., lot number: W10D10B104178, purity greater than 98%) was dissolved in methanol to prepare a 20. Mu.g/mL control solution.

Establishing a database of relevant chemical components of the DongDang angelica, analyzing mass spectrum fragments and 3 reference substance information by using a high performance liquid chromatography-time-of-flight mass spectrometry technology, and carrying out qualitative identification on the chemical components of the DongDang angelica extract extracted in the embodiment 1. The detection instrument is Triple TOF 5600 type high resolution mass spectrum (ABSCIEX corporation of America); the chromatographic conditions are as follows: xunonC 18 chromatographic column (2.1 mm 100mm,2.8 μm), gradient elution (0-2 min,85% A; 2-10 min, 85-63% A; 10-15 min, 63-50% A; 15-24 min, 50-30% A; 24-26 min, 30-20% A) with 0.1% formic acid water (A) -acetonitrile (B) as mobile phase, flow rate of 0.2mL/min, sample injection amount of 3 μL, ion source temperature of 550 ℃, electrospray ion source, detection in positive/negative ion mode, spray voltage-4500/+5500 kV, declustering voltage of 100V, collision energy of 40eV, scanning range m/z 80-1000, detection mode of IDA (information correlation acquisition mode). The methanol, acetonitrile and the like are all chromatographic purities, and the water for experiments is ultrapure water.

The mass spectrograms of 3 reference substances including the myrosin, the xanthotoxin and the protocatechuic acid are shown in figure 8. The results of the component identification of the extract of Donggui as extracted in example 1 are shown in Table 1, and 31 relevant components were identified in total.

TABLE 1 identification of Components of DongDang Gui extract of example 1

/>

/>

Then, to compare the differences between the chemical components of the DongDang extract obtained by different extraction methods, a Triple TOF 5600 type high resolution mass spectrum (ABSCIEX corporation in the United states) was also used to detect the contents of three effective components of geraniin, xanthotoxin and protocatechuic acid in the DongDang extract obtained by example 1 (abbreviated as sample 1) and the DongDang extract obtained by comparative example 1 (abbreviated as sample 2), and the chromatographic conditions are: the stationary phase is XunonC 18 chromatographic column (2.1 mm. Times.100 mm,2.8 μm), 0.1% formic acid water (A) -acetonitrile (B) are used as mobile phase for gradient elution (0-2 min,85% A; 2-10 min, 85-63% A; 10-15 min, 63-50% A; 15-24 min, 50-30% A; 24-26 min, 30-20% A), the flow rate is 0.2mL/min, the sample injection amount is 3 mu L, the ion source temperature is 550 ℃, the electrospray ion source is detected under positive/negative ion mode, the spray voltage is-4500/+5500 kV, the cluster removal voltage is 100V, the collision energy is 40eV, and the scanning range m/z is 80-1000. The total ion extraction flow diagrams of sample 1 and sample 2 are shown in fig. 9, wherein fig. 9 (A, C) is the total ion extraction flow diagram of sample 1, and fig. 9 (B, D) is the total ion extraction flow diagram of sample 2. The quantitative results of the three effective components in the sample 1 and the sample 2 are shown in the table 2, and the content of protocatechuic acid and the geraniin in the sample 1 is obviously higher than that in the sample 2 (P is less than 0.01), the content of the xanthotoxin in the sample 1 is 11.70+/-2.21 mug/g, and no xanthotoxin is detected in the sample 2. Example 1 and comparative example 1 differ only in that: the polystyrene-divinylbenzene macroporous resin chromatographic extract is adopted in example 1, and the methacrylate macroporous resin chromatographic extract is adopted in comparative example 1, which fully demonstrates that the method adopting the polystyrene-divinylbenzene macroporous resin chromatographic extract can pertinently extract and obtain the core component for resisting skin aging in the angelica sinensis.

Table 2 3 component contents of the two extracts of eastern angelica (μg/g,n＝6)

sample of	Protocatechuic acid	Pelargonium graveolens glycoside	Zanthoxylum bungeanum toxin
				Sample 1	16.37±1.23	42.15±7.62	11.70±2.21
Sample 2	1.51±0.24	1.63±0.45	Not detected

3. Application of Donggui extract

The extract of Donggui as obtained in example 1 was used to prepare essence and the effect of the prepared essence on aged skin was investigated.

Application example 1

The formulation composition of the essence containing the extract of DongDang Gui is shown in Table 3 below:

TABLE 3 formulation composition of essence of application example 1

The preparation method comprises the following steps:

soaking the hydrolyzed zinc hyaluronate and the hydrolyzed calcium hyaluronate with water, fully swelling for 12 hours, adding the soaked zinc hyaluronate and the hydrolyzed calcium hyaluronate into the water, stirring and dispersing uniformly, sequentially adding other components except polyhexamethylene biguanide and p-hydroxyacetophenone, stirring at the speed of 500r/min for 30 minutes at the temperature of 85 ℃, cooling to 40 ℃, adding polyhexamethylene biguanide and p-hydroxyacetophenone, and continuously stirring at the speed of 500r/min for 15 minutes.

The homogenizing and emulsifying equipment used in the embodiment is a FV-30 LFLISCO vacuum homogenizing and emulsifying machine manufactured by Shanghai Froude fluid machinery manufacturing Co., ltd, and has the functions of homogenizing, stirring, temperature control and the like.

Comparative example 1 was used

The formulation composition of the essence of the comparative application example is shown in the following table 4:

table 4 formulation composition of the serum using comparative example 1

Raw material name	Additive amount (%)	Manufacturer(s)
			Cyclohexylglycerol	5	ADEKA Corp.
Butanediol (butanediol)	3	Japanese Zhaoyao electric Co Ltd
			Hydrolysis of zinc hyaluronate	0.05	BLOOMAGE BIOTECH Co.,Ltd.
Hydrolyzed calcium hyaluronate	0.05	BLOOMAGE BIOTECH Co.,Ltd.
			N-acetylneuraminic acid	1	WUHAN ZHONGKE OPTICS VALLEY GREEN BIOTECHNOLOGY Co.,Ltd.
Ethylene diamine tetraacetic acid disodium salt	0.05	Suzhou Hua Xinxu chemical engineering Co., ltd
			Para hydroxy acetophenone	0.5	Shaanxi Xiazhou Biotech Co Ltd
HY-1 acrylic acid polymer resin	0.2	Sumitomo Seika Co., Ltd.
			Polyhexamethylene biguanide	0.4	Dragon sand group
Water and its preparation method	Allowance of	Child haha purified water

The preparation method comprises the following steps:

soaking the hydrolyzed zinc hyaluronate and the hydrolyzed calcium hyaluronate with water, fully swelling for 12 hours, adding the soaked zinc hyaluronate and the hydrolyzed calcium hyaluronate into the water, stirring and dispersing uniformly, sequentially adding other components except polyhexamethylene biguanide and p-hydroxyacetophenone, stirring at the speed of 500r/min for 30 minutes at the temperature of 85 ℃, cooling to 40 ℃, adding polyhexamethylene biguanide and p-hydroxyacetophenone, and continuously stirring at the speed of 500r/min for 15 minutes.

Then, in order to investigate the effect of the essence containing the extract of DongDang Gui on aging skin of the present invention, the anti-aging effect of the essence containing the extract of DongDang Gui prepared in application example 1 was verified at the human body level. The experimental procedure was as follows:

1. subject inclusion criteria

(1) Healthy female subjects aged 55-70 years, without clear systemic organic disease;

(2) Subjects were willing to use no other skin care, cosmetics, and receive other facial treatments (e.g., drugs, chemical exfoliations, lasers, etc.) throughout the study;

(3) The whole experiment is completed according to the experiment requirement during the whole study period, and the follow-up can be completed according to the requirement; (4) Informed consent was signed and willing to complete the study procedure.

2. Subject exclusion criteria

Allergic or serious skin allergy history to the essence containing the DongDang extract of the invention; patients who cannot be re-diagnosed on time or self-use other hormone-containing drugs during treatment; pregnant or lactating women.

3. General data of subjects

The study was co-included in 120 healthy female subjects, and subjects were randomized into 2 groups, treatment and control groups of 60 each, using a random number table method. The ages in the control group were 55-65 years, the average age (58.86 + -7.12) years, the treatment group was 54-66 years, and the average age (59.28 + -7.69) years. The two groups of patients were comparable with no significant differences in age comparison.

4. Experimental method

The right cheek of the subject is selected as a tested area by the treatment group, the essence containing the angelica sinensis extract prepared in the embodiment 1 is smeared in a looping mode after the skin is cleaned every day, the allergic parts such as the canthus and the like are avoided, each time is 2g in the morning and evening, the treatment is carried out for 28 days, 1 treatment course is carried out, and other similar products cannot be replaced or used in the treatment course.

The control group also selects the cheek on the right side of the subject as a tested area, the essence which is prepared in comparative example 1 and does not contain the angelica sinensis extract is smeared in a looping way after the skin is cleaned every day, the allergic parts such as the canthus and the like are avoided, each time is 2g in the morning and evening, the treatment is carried out for 28 days, and 1 treatment course is carried out in total, and other similar products cannot be replaced or used in the period.

During the trial, researchers suggested the subjects to change lifestyle and behavior patterns, such as ensuring adequate sleep, giving up smoking, giving up alcohol, avoiding eating spicy, cold, greasy foods, avoiding dust irritation and strenuous exercise, and preventing insolation under burning sun.

5. Observation index

(1) Skin physiological function index detection

Researchers measured the Skin gloss of the subject's facial Skin using a Skin gloss tester from the company kurage Khazaka, skin in-Glossymeter GL200, germany, 14 days, 28 days before, and measured the Skin elasticity of the subject using a Cutometer Skin elasticity tester from the company kurage Khazaka, germany, respectively. The test environment is a special skin function test room without ventilation, direct sunlight and relative humidity of 45-55% at the room temperature of 22-24 ℃. The subjects were tested after resting in the skin function test room for 30min to fit the environment without any topical facial medications and cosmetics on the day of testing, with regular facial cleansing. The skin gloss and elasticity test method is specifically as follows:

skin gloss: the skin gloss of the right cheek skin was measured using a skin gloss tester and was measured 5 times in succession, and an average value was taken. The larger the measurement, the more shiny the skin; conversely, the darker the skin.

Skin elasticity: the right eye corner was measured using a skin elasticity tester, and the measurements were performed 3 times in succession, and an average value was obtained. The parameters R2, r2=ua/Uf are selected, ua is the total rebound quantity of the skin from the elimination of the negative pressure to the next continuous test of the skin surface and then the negative pressure, uf is the maximum stretching quantity of the skin when the negative pressure exists, and the closer the R2 value is to 1, the better the skin elasticity is represented.

(2) Subjective evaluation of the subject

Subjects were collected in the form of a questionnaire and scored for their skin condition at 14 days and 28 days of use of the sample prior to use of the sample (day 0). The evaluation index includes skin dryness, glossiness, dark yellow, firmness, elasticity, and fine wrinkles. Each parameter was evaluated in terms of its subjective perception from 0 point (difference) to 9 points (good), and the degree of improvement was evaluated by using the difference between the indexes before and after the sample, with the evaluation criterion of i: no improvement (difference less than or equal to 0 point); II: slightly improving (0 score < difference less than or equal to 1 score); III: moderately improving (1 score < 2 score of difference); IV: obviously improves (the difference value is more than 2 and less than or equal to 3 minutes); v: significantly improved (difference > 3 points).

6. Adverse reactions

The adverse reaction symptoms, such as facial allergy, and the physical signs of the subjects before and after treatment are observed through inquiry or review.

7. Statistical method

The data are checked by two persons and then are input into SPSS 25.0 software for statistical analysis, the metering data are expressed by mean ± standard deviation(s), independent sample t test is adopted for inter-group comparison, and paired t test is adopted for intra-group comparison. Clinical efficacy is evaluated as grade data expressed in terms of frequency and frequency, and analyzed by rank-sum test, and differences are considered statistically significant when P < 0.05.

8. Results

(1) Comparison of skin physiological function detection indexes before and after treatment of subjects

The results of comparing the skin physiological function detection indexes before and after the treatment of the subjects are shown in table 5. The data in Table 5 show that there was no statistical difference in skin gloss, canthus skin elasticity R2 values (P > 0.05) for the 2 groups of patients before treatment began. Compared with the prior treatment, the skin glossiness and the skin elasticity R2 value of the canthus are obviously increased in the treatment groups at 14 days and 28 days after the treatment, have statistical difference (P <0.05 or P < 0.01), and are obviously better than the control groups (P <0.05 or P < 0.01) at 14 days and 28 days after the treatment.

TABLE 5 physiological functions of the skin of a subject before and after treatmentDetection index comparison/>

Note that: ﹡ P <0.05, ﹡ ﹡ P < 0.01 compared to pre-treatment; compared with the control group, deltaP is less than 0.05, deltaP is less than 0.01.

(2) Subjective evaluation statistics of subject

The statistical results of subjective evaluations of the subjects are shown in table 6. As can be seen from table 6, the subjects in the control group reached significant improvement in the skin dryness, glossiness, dark yellow, firmness, elasticity, and fine wrinkles at 14 days after using the essence without the extract of eastern angelica, at 1.67%, 0.00%, and 0.00%, respectively, and reached significant improvement at 0.00%. After 28 days of continued use, the significant improvement rates were 11.67%, 1.67%, 5.00%, 25.00%, 16.67% and 3.33%, respectively, and the significant improvement rates were 3.33%, 0.00%, 1.67%, respectively.

After the subjects in the treatment group use the essence containing the Donggui extract, each evaluation index is improved to different degrees, and the number of people with improved indexes is increased along with the prolonged use time. The subjects achieved significant improvements in skin dryness, gloss, darkness, firmness, elasticity, and fine wrinkles at 14 days of use of the samples at 66.67%, 5.00%, 20.00%, 40.00%, 33.33%, and 15.00%, respectively, and at 6.67%, 1.67%, 5.00%, 0.00%, 1.67%, and 1.67%, respectively. After 28 days of continued use, the subjects had further improved skin dryness, gloss, darkness, firmness, elasticity, and the proportion of significant improvement, respectively 48.33%, 51.67%, 50.00%, 61.67%, 33.33%, 43.33%, and significant improvement, respectively 35.00%, 25.00%, 15.00%, 10.00%, 25.00%, and 20.00%. Comparing the ratio of the treatment group to the control group to achieve the significant improvement, it can be seen that the ratio of the treatment group to achieve the significant improvement is significantly better than the control group.

Table 6 subject subjective evaluation statistics (n=60)

A comparison of photographs of subjects in a typical treatment group before and after 28 days of treatment is shown in fig. 10, in which the left side is a photograph before treatment and the right side is a photograph after 28 days of treatment, and as can be seen from fig. 10, the subjects have significant improvements in skin dryness, glossiness, darkness, firmness, elasticity and fine wrinkles after 28 days of treatment with the essence containing the extract of angelicae gigantis radix of the present invention.

(3) Adverse reactions

After 28 days of treatment and observation, no adverse reaction occurs, all subjects in the group complete the scheme treatment of the respective treatment course, the compliance is good during the whole study period, and the study process does not fall off.

9. Conclusion(s)

Clinical test results show that the essence containing the angelica sinensis extract can obviously improve the glossiness and the skin elasticity of the skin of a subject. From the subjective evaluation results of the subjects, the subjects showed that the product had various degrees of improvement on skin dryness, glossiness, dark yellow, firmness, elasticity and fine wrinkles after using the essence containing the extract of Donggui. Therefore, the essence containing the DongDang extract has good effect of resisting facial skin aging.

What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

Claims (10)

1. The extraction method of the anti-skin-aging angelica sinensis extract is characterized by comprising the following steps of:

(1) Pulverizing radix Angelicae sinensis, adding into 70% ethanol solution at a feed-liquid ratio of 1:10, pressurizing at 300MPa for 3min, vacuum filtering to obtain filtrate, centrifuging the filtrate, and collecting supernatant;

(2) Carrying out ultrafiltration treatment on the obtained supernatant, wherein a hollow cellulose acetate membrane is adopted as an ultrafiltration membrane, and ultrafiltrate is obtained after ultrafiltration is completed;

(3) Concentrating the obtained ultrafiltrate under reduced pressure to relative density of 1.15-1.25 to obtain DongDang radix Angelicae sinensis concentrated solution, filtering to obtain filtrate, subjecting the filtrate to chromatography with chromatography column containing polystyrene-divinylbenzene macroporous resin, eluting with water until the first eluent is neutral or sugar-free reaction is detected, eluting with eluent, and collecting the second eluent; the eluent consists of the following components in percentage by weight: acetic acid 2%, ethanol water solution 98% with volume concentration of 75%;

(4) Concentrating the second eluent under reduced pressure to recover ethanol to obtain extract, and freeze-drying to obtain anti-skin aging Donggui extract.

2. The method for extracting skin aging resistant Donggui extract according to claim 1, wherein in step (1), donggui is pulverized into fine powder having a particle size of 40 mesh.

3. The method for extracting an anti-skin aging extract of Donggui as claimed in claim 1 or 2, wherein in the step (1), the rotational speed of centrifugation is 5000r/min for 20min.

4. The method for extracting an anti-skin aging extract of angelicae gigantis radix of claim 1 or 2, wherein in the step (2), the hollow cellulose acetate membrane has a molecular weight of 50kD.

5. The method for extracting an anti-skin aging extract of Donggui as claimed in claim 1 or 2, wherein in the step (3), the Donggui concentrate is filtered with a 50 mesh sieve.

6. The method for extracting an anti-skin aging extract of angelicae gigantis radix of claim 1 or 2, wherein in the step (3), the flow rate of water elution is 2BV/h, and the flow rate of eluent elution is 2BV/h.

7. An anti-skin aging extract of Donggui as claimed in any one of claims 1 to 6.

8. The use of an extract of Donggui as claimed in claim 7 for the preparation of cosmetics.

9. An essence containing an anti-skin-aging angelica sinensis extract is characterized by comprising the following raw materials in parts by weight:

the skin aging resistant extract of Donggui as claimed in claim 7, wherein the extract comprises 2.5% of the extract of Donggui as defined in claim 7, 5% of cyclohexylglycerol, 3% of butylene glycol, 0.05% of hydrolyzed zinc hyaluronate, 0.05% of hydrolyzed calcium hyaluronate, 1% of N-acetylneuraminic acid, 0.05% of disodium edetate, 0.5% of p-hydroxyacetophenone, 0.2% of HY-1 acrylic acid poly resin, 0.4% of polyhexamethylene biguanide and the balance of water.

10. The method for preparing the essence containing the anti-skin-aging angelica sinensis extract as claimed in claim 9, which is characterized by comprising the following steps:

soaking zinc hyaluronate and calcium hyaluronate in water, fully swelling for 12h, adding water, dispersing uniformly, sequentially adding other components except polyhexamethylene biguanide and p-hydroxyacetophenone, stirring at a speed of 500r/min for 30min at 85 ℃, cooling to 40 ℃, adding polyhexamethylene biguanide and p-hydroxyacetophenone, and continuously stirring at a speed of 500r/min for 15 min.