A cornus officinalis fruit extract rich in morroniside and its preparation method and application

Abstract

The present invention provides a cornus officinalis fruit extract rich in morroniside and a preparation method and application thereof, and relates to the technical field of plant natural products. The cornus officinalis fruit extract rich in morroniside comprises morroniside, strychnoside and swertiaside, wherein the content of morroniside is 10% to 12%. The cornus officinalis fruit extract rich in morroniside of the present invention has excellent anti-inflammatory effect, can promote skin firmness and reduce wrinkles, and can be widely used in the preparation of cosmetics and skin care products. The preparation method of the cornus officinalis fruit extract rich in morroniside of the present invention efficiently extracts the active components in the cornus officinalis fruit by ethanol extraction and enzymatic hydrolysis treatment, and obtains the cornus officinalis fruit extract enriched with morroniside by combining column chromatography. The method is simple in process and has strong operability.

Description

Technical Field

[0001] The invention relates to the technical field of plant natural products, and in particular to a cornus officinalis fruit extract rich in morroniside and a preparation method and application thereof. Background Art

[0002] Iridoid compounds are a class of organic compounds with dual biological activities. They not only play a defensive role against certain plant species, such as the defensive response of iridoid glycosides in Rehmannia plants against Fusarium oxysporum, but also have a variety of pharmacological effects on animals, such as protective effects on the nervous system, anti-hepatotoxicity, hypoglycemic, anti-inflammatory, anti-spasmodic, anti-tumor, anti-viral, immunomodulatory and laxative biological activities, and are the active ingredients of many traditional Chinese medicines. Due to the special structure of iridoid compounds, they cannot be mass-produced by chemical synthesis, so extraction from plants is the main way to obtain these compounds.

[0003] Cornus officinalis Sieb. et Zucc., CO, is a plant of the genus Cornus in the family Cornaceae. It is also known as cornus, Shuzao, Shizao, Rouzao, Yulu, Zaopi, Yaozao, etc. Cornus officinalis is a deciduous or small tree species of the family Cornaceae. Its fruit is dry and ripe, and can be used as medicine. It is soft in texture and tastes slightly spicy, slightly bitter, and slightly hot. In China, the resources of Cornus officinalis are widely distributed, mainly along the Yangtze River and the Yellow River, and are mainly distributed in Henan, Shaanxi, Zhejiang, Anhui, Shanxi, Sichuan and other places. According to the Chinese Pharmacopoeia, the fruit is harvested when the first peel turns red in late autumn, and it is baked over a low fire or slightly scalded in boiling water, and the pit is removed in time and dried; Cornus officinalis contains a variety of iridoid components (such as morroniside, loganin, etc.) and the content is relatively rich.

[0004] Morroniside is a type of iridoid terpenoid compound, which is found in plants of the genera Sambucus, Patrinia, and Cornus. Research on morroniside involves many technical fields: studies have found that morroniside can reduce oxidative stress and apoptosis of nerve cells, and also promote the proliferation and migration of nerve cells. Further studies have found that it has the effect of improving nerve pain. As a potential drug molecule, morroniside has an anti-inflammatory effect in the treatment of liver and heart diseases by reducing the release of inflammatory factors; at the same time, in the treatment of diabetes, it has the effect of reducing glycation reactions and endothelial cell migration, and also has the effect of lowering blood sugar by regulating sugar intake; in the treatment of bone-related diseases, it promotes the proliferation of bone marrow cells to inhibit the apoptosis of bone cells.

[0005] In the field of dermatology and cosmetics, iridoid compounds are considered to be a type of soothing ingredient with comprehensive advantages due to their good water solubility and significant anti-inflammatory effects. As a relatively distinctive iridoid compound, the current research results of morroniside in the field of dermatology and cosmetics are still focused on anti-inflammatory and cell proliferation, while the research at the dermatology level only involves scalp hair loss prevention, and it still has broad development and application prospects. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention discloses a morroniside-rich Cornus officinalis fruit extract and a preparation method and application thereof. The morroniside content in the morroniside-rich Cornus officinalis fruit extract of the present invention reaches more than 10%, showing a significant effect of increasing skin firmness and reducing wrinkles.

[0007] In order to achieve the above technical objectives, on the one hand, the present invention provides a Cornus officinalis fruit extract rich in morroniside, wherein the Cornus officinalis fruit extract rich in morroniside comprises morroniside, strychnoside and swertiaside, wherein the content of morroniside is 10% to 12%.

[0008] The research and development team of the present invention has found through a large number of exploratory experiments and analyses that the Cornus officinalis fruit extract rich in morroniside in the above technical solution can play a firming and anti-wrinkle effect by participating in the degradation process of collagen, the activation process of matrix metalloproteins, and retinoic acid metabolism and inflammatory factor-related pathways. The Cornus officinalis fruit extract rich in morroniside can be applied to anti-inflammatory and / or anti-aging cosmetics or skin care products, which is not disclosed in the prior art.

[0009] Furthermore, the content of strychnoside in the Cornus officinalis fruit extract rich in morroniside is 10%-30%, and the content of swertiain is 2%-6%. The embodiment of the present invention shows the anti-wrinkle effect of the Cornus officinalis fruit extract rich in morroniside.

[0010] Further, the pH range of the Cornus officinalis fruit extract rich in morroniside is 2.5 to 6.5;

[0011] Furthermore, the relative density of the Cornus officinalis fruit extract rich in morroniside is 1.025-1.125.

[0012] On the other hand, the present invention provides a method for preparing a cornus officinalis fruit extract rich in morroniside, the method comprising:

[0013] (1) adjusting the pH value of the ethanol solution to a range of 4 to 5, adding Cornus officinalis fruit, and mixing evenly to obtain a mixture to be extracted;

[0014] (2) adding a composite enzyme comprising pectinase and cellulase to the mixture to be extracted, and performing gradient temperature enzymolysis; after the enzymolysis is completed, the crude extract is obtained by inactivating the enzyme, separating and concentrating; the gradient temperature enzymolysis comprises maintaining the temperature at 45-55° C. for 0.5-1.5 h, and then maintaining the temperature at 65-75° C. for 0.5-1.5 h;

[0015] (3) The crude extract is subjected to column chromatography to obtain the Cornus officinalis fruit extract rich in morroniside.

[0016] The above technical solution is beneficial to improving the extraction rate of active components of Cornus officinalis fruit by extracting and separating with ethanol and combining it with specific enzymatic hydrolysis to decompose the cell wall; in addition, the above technical solution can obtain a crude Cornus officinalis fruit extract with a higher morroniside content by adjusting the pH value of the system and using a gradient temperature for enzymatic hydrolysis, and further purification by column chromatography allows the technical solution of the present invention to obtain a Cornus officinalis fruit extract with a morroniside content of more than 10%.

[0017] In a further example of the present invention, the concentration of the ethanol solution can be selected from 60% to 80%, and 60%, 65%, 70%, 75%, and 80% can be selected, but it is not limited to the listed range. Other unlisted values ​​within the above numerical range are also applicable to the present invention. The appropriate ethanol concentration helps to break the cell wall of the cornus fruit, making it easier to dissolve the active components contained therein. In some optional examples of the present invention, the concentration of the ethanol solution can be selected as 60%. It should be noted that the concentration of the ethanol solution of the present invention is a volume fraction concentration.

[0018] In a further example of the present invention, the solid-liquid ratio of ethanol to cornus fruit can be selected as 1: (8~15), such as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, but is not limited to the listed ranges. Other unlisted values ​​within the above numerical ranges are also applicable to the present invention. By optimizing the amount of ethanol and cornus fruit, the extraction efficiency of active components in cornus fruit can be improved, and the input of ethanol can be reduced. In some optional examples of the present invention, the solid-liquid ratio of ethanol to cornus fruit can be selected as 1:10. It should be noted that the solid-liquid ratio described in the present invention is a mass-to-volume ratio; the ethanol solution used in the present invention can be recovered and recycled in subsequent separation operations.

[0019] In a further example of the present invention, the added amount of the complex enzyme can be selected to be 0.3%~0.5% of the mass of the Cornus officinalis fruit, and can be selected from 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, but is not limited to the listed range. Other unlisted values ​​within the above numerical range are also applicable to the present invention. Adding an appropriate amount of the complex enzyme can effectively decompose the cell wall and improve the extraction efficiency.

[0020] In a further example of the present invention, the mass ratio of pectinase to cellulase in the composite enzyme can be selected as 1: (1-3), such as 1: 1, 1: 1.2, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.8, 1: 2, 1: 1.22, 1: 1.24, 1: 1.25, 1: 1.26, 1: 1.28, 1: 3, but not limited to the listed range, other unlisted values ​​within the above numerical range are also applicable to the present invention; based on a large amount of experimental data, it is found that composite enzymes with a specific ratio can play a better enzymatic effect in the extraction of Cornus officinalis fruit. In an optional example of the present invention, the mass ratio of pectinase to cellulase in the composite enzyme is 1: 1.

[0021] In a further example of the present invention, the temperature of the enzyme inactivation operation can be selected to be 85~95°C, and can be 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, and 95°C, but is not limited to the listed range, and other values ​​not listed in the above numerical range are also applicable to the present invention; the enzyme inactivation time can be selected to be at least 20min, and can be 20min, 25min, 30min, 35min, 40min, 45min, 50min, and 60min, but is not limited to the listed range, and other values ​​not listed in the above numerical range are also applicable to the present invention. In the present invention, the enzyme inactivation operation is performed by raising the system temperature, and the enzyme activity can be quickly and efficiently terminated without the addition of additional chemical reagents. At the same time, it can also prevent the effective ingredients in the extract from being further decomposed by the enzyme, and the operability is strong.

[0022] In a further example of the present invention, the separation operation in step (2) can be performed at 40-50°C, and can be 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, or 50°C, but is not limited to the listed range. Other values ​​not listed within the above numerical range are also applicable to the present invention. In the actual process, after the enzyme inactivation operation is completed, the temperature of the system can be reduced to 40-50°C to maintain the activity of the components in the extract. The filtrate can be collected by centrifugation and the filter residue can be discarded.

[0023] In a further example of the present invention, the concentration operation may be selected as concentrator concentration or rotary evaporation concentration. In the specific operation process, the filtrate obtained by the separation operation is added to the concentrator for concentration to obtain a supernatant, and the supernatant is the crude extract. A person skilled in the art may select to concentrate the filtrate obtained by the separation operation in batches, for example, 3 to 5 times to improve the efficiency of the concentration operation. In actual operation, the number of batch concentrations may be selected as needed.

[0024] In a further example of the present invention, the step (3) is purified by polyamide chromatography column, the sample loading flow rate is 0.5-10 times column volume / hour, 0.5-10 times column volume of purified water is eluted, the elution flow rate is 0.5-10 times column volume / hour, and the purified water eluate is collected and concentrated at the beginning of sample loading to obtain the Cornus officinalis fruit extract rich in morroniside.

[0025] It should be noted that the present invention also includes preliminary processing of the cornus fruit medicinal material. Specifically, the cornus fruit medicinal material can be dried and crushed into powder for later use.

[0026] The embodiment of the present invention shows that the content of morroniside in the cornus officinalis fruit extract prepared by the above method for preparing the cornus officinalis fruit extract rich in morroniside is above 10%. Further exploratory experiments of the present invention verify its excellent technical effects of anti-inflammatory, promoting skin firmness and reducing wrinkles.

[0027] On the other hand, the present invention provides the use of the above-mentioned Cornus officinalis fruit extract rich in morroniside and the Cornus officinalis fruit extract rich in morroniside prepared by the above-mentioned preparation method in the preparation of cosmetics or skin care products.

[0028] Furthermore, the cosmetics or skin care products are anti-inflammatory and / or anti-aging cosmetics or skin care products.

[0029] Furthermore, the cosmetic or skin care product is a cosmetic or skin care product for preventing or improving wrinkles.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the morroniside-rich cornus fruit extract of the present invention has a better anti-inflammatory effect than pure morroniside, and can promote skin firmness and reduce wrinkles, and can be widely used in the preparation of cosmetics and skin care products. The preparation method of the morroniside-rich cornus fruit extract of the present invention efficiently extracts the active components in the cornus fruit by ethanol extraction and enzymatic hydrolysis, and combines column chromatography to obtain the morroniside-enriched cornus fruit extract; the preparation method of the present invention has a simple process and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1 The 210nm ultraviolet absorption graph of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention is shown;

[0033] Figure 2 The 254nm ultraviolet absorption graph of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention is shown;

[0034] Figure 3 The intersection Venn diagram of the firming and anti-wrinkle target points of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention is shown;

[0035] Figure 4The interaction network diagram of the target proteins of the firming and anti-wrinkle of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention is shown; wherein, the full name of RARA is Retinoic acid receptor alpha, and the full name in Chinese is retinoic acid receptor A; the full name of MMP9 is Matrix metalloproteinase-9, and the full name in Chinese is matrix metalloproteinase 9; the full name of ELANE is Neutrophil elastase, and the full name in Chinese is neutrophil elastase; the full name of MMP1 is Matrix metalloproteinase-1, and the full name in Chinese is matrix metalloproteinase 1; the full name of TP53 is Cellular tumorantigen p53, and the full name in Chinese is cellular tumor antigen p53; the full name of MMP2 is Matrix metalloproteinase-2, and the full name in Chinese is matrix metalloproteinase 2; the full name of MMP12 is Matrix metalloproteinase-12, and the full name in Chinese is matrix metalloproteinase 12; the full name of MMP13 is Matrix metalloproteinase-13, and the full name in Chinese is matrix metalloproteinase 13; the full name of SIRT1 is NAD-dependent protein deacetylase Sirtuin-1, the full name in Chinese is nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1; RARB, the full name is Retinoic acid receptor beta, the full name in Chinese is retinoic acid receptor B; RXRG, the full name is Retinoic acid receptor RXR-gamma, the full name in Chinese is retinoic acid receptor RXR-G; CTRC, the full name is Chymotrypsin-C, the full name in Chinese is chymotrypsin C; P2RX7, the full name is P2X purinoceptor 7, the full name in Chinese is P2X purine receptor 7; GLB1, the full name is Beta-galactosidase, the full name in Chinese is β-galactosidase; GAPDH, the full name is Glyceraldehyde-3-phosphate dehydrogenase, the full name in Chinese is glyceraldehyde-3-phosphate dehydrogenase; PTGS2, the full name is Prostaglandin G / Hsynthase 2, the full name in Chinese is prostaglandin G / H synthase 2; PTPN2, the full name is Tyrosine-protein phosphatase non-receptor type 2, the full name in Chinese is tyrosine protein phosphatase non-receptor type 2; RARG’s full name is Retinoic acid receptor gamma, the full name in Chinese is retinoic acid receptor G;HSD11B2 stands for 11-beta-hydroxysteroid dehydrogenase type 2; NR3C2 stands for Mineralocorticoid receptor; HRPT1 stands for Hypoxanthine Phosphoribosyltransferase 1;

[0036] Figure 5 The figure shows the result of GO enrichment analysis of the Cornus officinalis fruit extract rich in morroniside prepared in Example 1 of the present invention;

[0037] Figure 6 The figure shows the result of Reactome pathway enrichment analysis of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention;

[0038] Figure 7 A comparative diagram showing the anti-inflammatory effects of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention and morroniside; Note: The hollow circles represent the number of sample repetitions (three hollow circles mean that the sample was repeated 3 times in the corresponding experiment), " " represents the comparison between negative control group and blank control group (" ” represents p<0.001), “#” represents the comparison between Example 1-Cornus officinalis extract-0.5% and pure morroniside of the same concentration (“#” represents p<0.05). From a statistical point of view, “#” and “ " indicates that the data have statistically significant differences; the blank control in the figure represents the normal cell group; the negative control represents the stimulated cell group, which shows an inflammatory state (lps: lipopolysaccharide); Example 1 and pure morroniside at the same concentration are negative controls, and different samples are added to the experimental groups;

[0039] Figure 8 The figure shows the comparison of the expression of elastin by the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention and morroniside; Note: The hollow circles represent the number of sample repetitions (three hollow circles mean that the sample was repeated 3 times in the corresponding experiment), " " and "#" represent significant analysis, ” represents comparison with blank control group and Example 1-Cornus officinalis extract-0.5% and pure morroniside of the same concentration (“*” represents p<0.05, “**” represents p<0.01; “***” represents p<0.001); error bars represent the mean ± mean square error of 3 repeated samples;

[0040] Fig. 9A comparison chart showing the effects of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention and morroniside on the expression of retinoic acid receptor RARB; Note: The hollow circles represent the number of sample repetitions (three hollow circles mean that the sample was repeated 3 times in the corresponding experiment), " " and "#" represent significant analysis, " represents the comparison with the blank control group and Example 1 - Cornus officinalis extract - 0.5% and the same concentration of pure morroniside (" " represents p < 0.05," " represents p < 0.01; " ” represents p<0.001); Error bars represent the mean ± mean square error of 3 repeated samples;

[0041] Fig.10 The figure shows the promoting effect of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention on TGF-β; Note: The hollow circles represent the number of sample repetitions (three hollow circles mean that the sample was repeated 3 times in the corresponding experiment), " " and "#" represent significant analysis, " represents the comparison with the blank control group and different concentrations of Example 1-Cornus officinalis extract (" " represents p < 0.05," " represents p < 0.01; " ” represents p<0.001); Error bars represent the mean ± mean square error of 3 repeated samples;

[0042] Fig.11 The figure shows the promoting effect of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 of the present invention on type III collagen; Note: The hollow circles represent the number of sample repetitions (three hollow circles mean that the sample was repeated 3 times in the corresponding experiment), " " and "#" represent significant analysis, " represents the comparison with the blank control group and different concentrations of Example 1-Cornus officinalis extract (" " represents p < 0.05," " represents p < 0.01; " ” represents p<0.001); Error bars represent the mean ± mean square error of 3 repeated samples. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form, i.e., not intended to limit the scope of protection of the present invention.

[0044] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods. Example

[0045] A method for preparing a cornus officinalis fruit extract rich in morroniside comprises the following steps:

[0046] (1) Weigh the cornus officinalis fruit medicinal material (origin: Qinling Mountains, Shaanxi Province), dry it, and grind it into powder for later use. Weigh 1 kg of 60% ethanol according to a solid-liquid ratio of 1:10, adjust the pH to 5, and then put it into an extraction tank; then add the cornus officinalis fruit powder, stir and mix evenly to obtain a mixture to be extracted.

[0047] (2) Add 0.5% of the weight of the dried cornus fruit pectinase and cellulase to the mixture to be extracted, wherein the mass ratio of pectinase to cellulase is 1:1; first, circulate and extract at a constant temperature of 50°C for 1 hour, then heat to 70°C for 1 hour; then heat to 90°C to inactivate the enzyme for 30 minutes to obtain a crude extract; then cool to 50°C and release the crude extract into a liquid storage barrel; centrifuge the crude extract, collect the filtrate and discard the filter residue; add the filtrate to a concentrator for concentration three times, and then centrifuge to obtain a crude extract. It should be noted that cyclic extraction in this embodiment refers to the operation of promoting full contact between the material and the solvent through stirring, shaking, etc.

[0048] (3) diluting the crude extract with pure water, adding it to a chromatography column filled with a special polyamide filler for chromatography, and then eluting it with pure water; adding the collected eluate to a concentrator for concentration three times, and then centrifuging to obtain a cornus officinalis fruit extract rich in morroniside. The cornus officinalis fruit extract rich in morroniside has a pH value of 2.5-6.5 and a relative density of 1.025-1.125.

[0049] (4) Three samples were prepared in parallel according to the same process conditions and named as samples 1-1, 1-2, and 1-3 respectively.

[0050] The cornus officinalis fruit extract rich in morroniside obtained in this embodiment can be used as a cosmetic grade raw material, but in the field of cosmetic raw materials, there is no clear regulation on the ingredients of cornus officinalis fruit. According to the requirements of the 2020 edition of the Chinese Pharmacopoeia, morroniside, one of the core components of cornus officinalis fruit extract, was calibrated as the main quality reference substance. Based on this, this embodiment detected the content of morroniside in the obtained cornus officinalis fruit extract; the specific determination method is as follows:

[0051] 1. Sample pretreatment: (1) Take 1 mL of sample, centrifuge for 10 min (13000 rpm, 4 ℃), take 20 μL of supernatant, add 180 μL of 50% methanol-water to dilute 10 times. (2) Take 10 μL of diluted supernatant, add 490 μL of 50% methanol-water to dilute 50 times again. (3) Use a syringe to draw 200 μL of supernatant, add internal standard (L-2-chlorophenylalanine), vortex for 10 s, transfer to LC injection vial, and store at -80 ℃ until LC-MS analysis.

[0052] 2. In this experiment, UPLC-ESI-MS / MS analysis method was used to qualitatively and quantitatively detect the target metabolites. The specific analysis conditions and analysis methods are as follows: (1) Chromatographic method includes injection volume: 5 μL; mobile phase: A (0.1% formic acid-water solution), B (acetonitrile); flow rate: 0.35 mL / min; (2) Gradient Elution Procedures: 0 min A / B (95:5, V / V), 1.0 min A / B (95:5, V / V), 3 min A / B (10:90, V / V), 4.5 min A / B (10:90, V / V), 4.51 min A / B (95:5, V / V), 5 min A / B (95:5, V / V); (3) Mass spectrometry method includes curtain gas: 55 (psi); collision-activated ionization (CII) dissociation, CAD) parameters: medium; positive ion spray voltage: 5500 V; negative ion spray voltage: -4500 V; ion source temperature: 500°C; column temperature: 40°C; spray gas (Gas1): 50 (psi); auxiliary heating gas (Gas2): 50 (psi).

[0053] It was determined that the morroniside content of sample 1-1 in the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 was 10%, the morroniside content of sample 1-2 was 10.9%, and the morroniside content of sample 1-3 was 11%.

[0054] Furthermore, this example tests the efficacy of the morroniside-rich Cornus officinalis fruit extract prepared in Example 1.

[0055] 1. Full ingredient analysis

[0056] The whole component analysis of the Cornus officinalis fruit extract rich in morroniside prepared in Sample 1-1 of Example 1 was performed by high-resolution liquid chromatography-mass spectrometry to identify other components besides morroniside. The specific analysis steps are as follows:

[0057] 1.1 Pre-treatment

[0058] The sample was taken out and evenly mixed, and 100 μL of the sample was taken into a 1.5 mL centrifuge tube; 400 μL of water (containing mixed internal standard, 4 μg / mL) was added, and vortexed for 1 min; ultrasonic extraction was performed in an ice water bath for 20 min; centrifugation was performed for 10 min (14000 rpm, 4°C); 200 μL of the supernatant was taken and placed into an LC-MS injection vial with an inner liner for analysis.

[0059] 1.2 Liquid chromatography-mass spectrometry conditions

[0060] The analytical instrument for this experiment is a liquid chromatography-mass spectrometry system consisting of an ACQUITY UPLCI-Class HF ultra-high performance liquid phase tandem QE high-resolution mass spectrometer.

[0061] Chromatographic conditions: Chromatographic column: ACQUITY UPLC H ST3 (100 mm × 2.1 mm, 1.8 um); Column temperature: 45 ° C;

[0062] Mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile; flow rate: 0.35mL / min; injection volume: 5μL.

[0063] PDA scanning range: 210-400 nm; elution gradient as shown in Table 1.

[0064]

[0065] Mass spectrometry conditions:

[0066] Ion source: HESI; sample mass spectrometry signal acquisition uses positive and negative ion scanning modes respectively. Data acquisition mode: DDA; Scanning mode: Full MS / dd-MS2 (TOP 8). The mass spectrometry parameter information table is shown in Table 2.

[0067]

[0068] In this example, the cornus officinalis fruit extract rich in morroniside prepared in Example 1 was subjected to full component analysis and identification, including: 1) by analyzing and measuring 210nm (the result is as follows Figure 1 ) and 254nm (results as Figure 2 ) was used to determine that the Cornus officinalis fruit extract contained saponins and basic structures such as benzene rings and double bonds based on its ultraviolet absorption under ultraviolet light. 2) Based on high-precision mass spectrometry analysis and self-built standard compound database and other technical platforms, the full components of Cornus officinalis fruit extract were qualitatively analyzed, and the relative contents of the core components strychnosin and swertinoside were obtained using peak area as a semi-quantitative basis (relative content basis).

[0069] 2. Network pharmacology analysis

[0070] 2.1 Target prediction

[0071] The chemical structures of the active ingredients of the above-mentioned compounds were collected in mol2 format and converted into sdf format files with the help of Open Babel 2.3.2 software. They were then imported into the SwissTargetPrediction (http: / / www.swisstargetprediction.ch) database and the attributes were set to "Homo sapiens" to obtain the prediction results of the potential targets of the full ingredients of Cornus officinalis fruit extract, and then the obtained target data were deduplicated and summarized.

[0072] 2.2 Exploration of anti-wrinkle targets and screening of core targets

[0073] Upload the data in item "1" and the internally sorted skin anti-wrinkle related targets to the online Venny 2.1.0 tool platform (https: / / bioinfogp.cnb.csic.es / tools / ven-ny / ) to obtain common targets and draw a Venn diagram (such as Figure 3 ).

[0074] 2.3 Construction of protein interaction network (PPI)

[0075] The STRING database (https: / / string-db.org) is an online database for searching known protein interactions. The target data of the intersection of Cornus officinalis fruit extracts were imported into it, and the biological species was set to "Homo sapiens" and the interaction score was >0.9 and the free nodes were hidden to construct a protein-protein interaction (PPI) network and download its tsv format file (the results are shown in Figure 4 ).

[0076] 2.4 GO and KEGG pathway enrichment analysis

[0077] The targets obtained from the intersection screening were input into the Metascape database for GO and KEGG functional enrichment analysis, and the analyzed data were visualized. Figure 5 , Figure 6 shown.

[0078] Combination Figure 3 , Figure 4, based on network pharmacology tools and an internally built skin target database, 21 targets related to skin firming and anti-wrinkle were finally found among the 573 targets matched by the cornus fruit extract, including targets related to collagen and elastin degradation such as MMP1, MMP2, MMP13, and MMP9, and targets related to retinol metabolism such as RARA, RARB, and RARG. From the target prediction analysis, it can be seen that the cornus fruit extract rich in morroniside of the present invention can show potential firming and anti-wrinkle effects, and show a vitamin A-like effect.

[0079] The 21 screened targets were then subjected to GO and Reactome functional enrichment analysis to identify the processes involved in regulation and associated pathways between targets. Figure 5 It can be seen that the GO enrichment analysis reflects that the cornus officinalis fruit extract rich in morroniside of the present invention is involved in biological processes such as retinoic acid metabolism, degradation of extracellular matrix, synthesis of collagen, etc.; participates in the synthesis of extracellular matrix including collagen; and participates in molecular functions such as matrix metalloproteinase activity and nuclear transcription receptor activity. Figure 6 As shown, the results of the firming and anti-wrinkle target pathway enrichment analysis show that the Cornus officinalis fruit extract rich in morroniside of the present invention can exert the firming and anti-wrinkle effect by participating in the degradation process of collagen, the activation process of matrix metalloproteins, and the retinoic acid metabolism and inflammatory factor-related pathways.

[0080] 3. In vitro cell anti-wrinkle efficacy test

[0081] Specifically, human primary fibroblasts in the exponential growth phase were prepared into single-cell suspensions, and the cells were inoculated on 6-well plates at a density of 1.0-1.2×105cells / mL. The final culture volume was 2 mL, and the cells were cultured at 37°C with a medium containing 10% fetal bovine serum until the cells adhered to the wall. The supernatant was discarded, and each well was replaced with freshly prepared test substances of different concentrations (serum-free). The cells were collected and RNA was extracted according to the standard operating procedures for RNA extraction. 1.2 μg was aliquoted and stored at -80°C for RNAseq. The remaining RNA was divided into two parts, one was reverse transcribed into cDNA for qPCR detection, and the other RNA was aliquoted and stored at -80°C. The expression of Col-III, Elastin, and TGF-β mRNA was detected by qPCR.

[0082] 3.1 Comparison of the efficacy of the cornus officinalis fruit extract rich in morroniside and morroniside in sample 1-1 of Example 1

[0083] 1) In this example, the anti-inflammatory effects of the cornus fruit extract rich in morroniside in Example 1 and morroniside at the same concentration were compared. Figure 7 As shown. Figure 7It can be confirmed that the anti-inflammatory effect (specifically, the inhibitory effect on the inflammatory factor IL-6) of the Cornus officinalis fruit extract of the present invention is unexpectedly better than that of pure morroniside, with an inhibition rate of 56.33%, while the inhibition rate of pure morroniside is 51.25%.

[0084] It should be noted that the same concentration in this example means that the concentration of morroniside in the morroniside-rich Cornus officinalis fruit extract prepared in Example 1 is consistent with the concentration of the morroniside pure sample. This phenomenon shows that the Cornus officinalis fruit extract prepared by the extract preparation process of the present invention retains the anti-inflammatory effect of morroniside, while other ingredients synergistically enhance the anti-inflammatory effect of morroniside.

[0085] 2) In this example, the anti-aging effects of the cornus officinalis fruit extract rich in morroniside in sample 1-1 of Example 1 and morroniside were compared. The results are as follows: Figure 8 , Fig. 9 shown.

[0086] Through preliminary exploratory experiments, it was found that the anti-aging effect of morroniside is limited. Figure 8 and Fig. 9 This finding was also verified: Morroniside did not show improvement effects on elastin Elastin and retinoic acid receptor RARB in fibroblasts, but the morroniside-rich Cornus officinalis fruit extract prepared in Sample 1-1 of Example 1 showed a relatively good improvement effect, with improvement rates of 106.8% (Elastin) and 552.2% (RARB), respectively. Therefore, the morroniside-rich Cornus officinalis fruit extract in Sample 1-1 of Example 1 of the present invention not only enriched the morroniside content and improved the anti-inflammatory effect of morroniside, but also achieved the anti-aging effect that morroniside itself did not have, and had a broader use value than simple morroniside.

[0087] 3.2 Improvement of ECM by the morroniside-rich Cornus officinalis fruit extract of the present invention

[0088] TGF-β (transforming growth factor-β) is one of the key regulators of collagen synthesis in the skin. It regulates the activity of fibroblasts through the classic SMAD signaling pathway and promotes the production of type I and type III collagen. Type I and type III collagen are important components of skin structure and can increase skin elasticity and firmness, thereby reducing the formation of wrinkles.

[0089] As an important target molecule for skin anti-aging and skin care, TGF-β improves skin smoothness and elasticity by promoting collagen production, inhibiting its degradation, and regulating cell proliferation and differentiation. This example found that the cornus fruit extract rich in morroniside of the present invention has a good promoting effect on TGF-β (such as Fig.10At the same time, the expression test results of downstream proteins regulated by TGF-β showed that the cornus fruit extract rich in morroniside can significantly promote the expression of type III collagen (COL-III) (as shown in Fig.11 as shown).

[0090] The above examples demonstrate that the morroniside-rich Cornus officinalis fruit extract of the present invention can improve and reshape the ECM structure by regulating the TGF-β signaling pathway, promote skin firmness, and reduce the occurrence of wrinkles, and can be widely used in the preparation of cosmetics or skin care products.

[0091] In addition, the present invention also explores the technical advantages of the preparation method of the Cornus officinalis fruit extract rich in morroniside by setting up Comparative Example 1.

[0092] Comparative Example 1

[0093] Comparative Example 1.1

[0094] A method for preparing a cornus officinalis fruit extract, wherein the raw materials and parameter control used in this comparative example preparation method are the same as those in Example 1, except that step (3) is not included. Three samples are prepared in parallel according to this process condition, and are named samples 1.1-1, 1.1-2, and 1.1-3, respectively.

[0095] Comparative Example 1.2

[0096] A method for preparing a cornus officinalis fruit extract. The raw materials and parameter control used in this comparative example preparation method are the same as those in Example 1, except that the complex enzyme is not added in step (2). Three samples are prepared in parallel according to this process condition, and are named samples 1.2-1, 1.2-2, and 1.2-3, respectively.

[0097] Comparative Example 1.3

[0098] A method for preparing a cornus officinalis fruit extract. The raw materials and parameter control used in this comparative example preparation method are the same as those in Example 1, except that an equal volume of pure water is used in step (1) instead of the ethanol solution. Three samples are prepared in parallel according to this process condition, and are named samples 1.3-1, 1.3-2, and 1.3-3, respectively.

[0099] Comparative Example 1.4

[0100] A method for preparing a cornus fruit extract specifically comprises the following steps: weighing cornus fruit medicinal materials (origin: Qinling Mountains, Shaanxi), drying, crushing into powder, and setting aside. Weighing 1 kg of pure water according to a material-liquid ratio of 1:10, adjusting the pH to 5, putting into an extraction tank, adding cornus fruit powder, stirring and mixing evenly; then cyclically extracting at a constant temperature of 50°C for 1 hour, and then heating to 70°C for 1 hour. Cooling to 40-50°C, releasing the crude extract into a liquid storage barrel. Centrifuging the crude extract, collecting the filtrate to obtain the cornus fruit extract. Three samples were prepared in parallel according to this process condition, and were named samples 1.4-1, 1.4-2, and 1.4-3, respectively.

[0101] The present invention measured the morroniside content (mass percentage) in the Cornus officinalis fruit extracts prepared in Example 1 and Comparative Example 1. The results are shown in Table 3.

[0102]

[0103] Note: “ns” means that morroniside was not detected in the extract.

[0104] Combining Example 1 and Comparative Examples 1.1-1.4, it can be verified that the present invention can achieve rapid enrichment of morroniside in the Cornus officinalis fruit extract by ethanol extraction combined with enzymatic treatment and chromatography column purification, and obtain a Cornus officinalis fruit extract with a higher morroniside content, reflecting that the technical solution of the present invention can obtain a higher extraction rate. In addition, combined with the full component analysis, it is found that in addition to morroniside, the morroniside content in the Cornus officinalis fruit extract prepared in the embodiment of the present invention is 10% to 30%, and the swertiain content is in the range of 2% to 6%.

[0105] It should be noted that the above contents are further detailed descriptions of the present invention in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple improvements can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A cornus officinalis fruit extract rich in morroniside, characterized in that: The cornus officinalis fruit extract rich in morroniside comprises morroniside, strychnine and swertiain, wherein the content of morroniside is 10% to 12%, the content of strychnine is 10% to 30%, and the content of swertiain is 2% to 6%; The cornus officinalis fruit extract rich in morroniside is prepared by the following method: (1) adjusting the pH value of the ethanol solution to a range of 4 to 5, adding Cornus officinalis fruit, and mixing evenly to obtain a mixture to be extracted; (2) adding a composite enzyme comprising pectinase and cellulase to the mixture to be extracted, and performing gradient temperature enzymolysis; after the enzymolysis is completed, the crude extract is obtained by inactivating the enzyme, separating and concentrating; the gradient temperature enzymolysis comprises maintaining the temperature at 45-55° C. for 0.5-1.5 h, and then maintaining the temperature at 65-75° C. for 0.5-1.5 h; The mass ratio of pectinase to cellulase in the complex enzyme is 1:(1-3); The added amount of the complex enzyme is 0.3% to 0.5% of the weight of the cornus fruit; (3) The crude extract is subjected to column chromatography to obtain the Cornus officinalis fruit extract rich in morroniside; The step (3) adopts polyamide chromatography column chromatography purification, the sample loading flow rate is 0.5 to 10 times column volume / hour, 0.5 to 10 times column volume of purified water elution, the elution flow rate is 0.5 to 10 times column volume / hour, and the purified water eluate is collected and concentrated at the beginning of sample loading to obtain the Cornus officinalis fruit extract rich in morroniside.

2. The cornus officinalis fruit extract rich in morroniside according to claim 1, characterized in that The pH range of the cornus officinalis fruit extract rich in morroniside is 2.5-6.5; the relative density of the cornus officinalis fruit extract rich in morroniside is 1.025-1.

125.

3. The cornus officinalis fruit extract rich in morroniside according to claim 1, characterized in that The concentration of the ethanol solution is 60% to 80%; And / or, the solid-liquid ratio of the ethanol solution to the Cornus officinalis fruit is 1:(8-15).

4. The cornus officinalis fruit extract rich in morroniside according to claim 1, characterized in that The temperature of the enzyme inactivation operation is 85-95°C, and the enzyme inactivation time is at least 20 minutes; And / or, the separation operation in step (2) is performed at 40-50°C; And / or, the concentration operation is concentrator concentration or rotary evaporation concentration.

5. Use of the morroniside-rich Cornus officinalis fruit extract according to any one of claims 1 to 4 in the preparation of anti-aging cosmetics.

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