CN116655820B - Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics - Google Patents

Abstract

The invention belongs to the field of natural product cosmetics application, and discloses vine tea acidic polysaccharide AGP-3a, an extraction and separation method thereof and application thereof in preparing anti-inflammatory cosmetics. The method comprises the following steps: dissolving the dried Ampelopsis grossedentata water extract with water, adding absolute ethyl alcohol, and obtaining Ampelopsis grossedentata crude polysaccharide by a water-soluble alcohol precipitation method; removing protein and pigment by Sevag method and active carbon to obtain Ampelopsis grossedentata crude polysaccharide; dissolving Ampelopsis grossedentata crude polysaccharide in ultrapure water, passing through DEAE-agarose gel CL-6B column, gradient eluting with NaCl, mixing and collecting 140-175 Guan Xituo th peak solution, dialyzing with 3.5kDa dialysis bag for 48 hr, concentrating under reduced pressure, and freeze drying to obtain Ampelopsis grossedentata polysaccharide; eluting with ultra-pure water by passing through Sephadex-G75 chromatographic column, mixing and collecting the eluting peak parts in the 9 th-30 th tube, concentrating under reduced pressure, and freeze drying to obtain vine tea acidic polysaccharide AGP-3a with uniform molecular weight.

Description

Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics

Technical Field

The invention belongs to the field of natural product cosmetics application, and in particular relates to vine tea acidic polysaccharide AGP-3a, an extraction and separation method thereof and application thereof in preparing anti-inflammatory cosmetics.

Background

Inflammatory response is a beneficial self-protecting defensive response of the body to irritation, usually caused by bacterial infection, chemical injury and environmental pollution. In the course of inflammation, macrophages in the innate immune system play an important role in combating pathogens, alleviating inflammation, and the like. When macrophages are stimulated, they release cytokines such as Nitric Oxide (NO), tumor necrosis factor (TNF-alpha), interleukin-6 (IL-6), and the like, which are associated with inflammation. Therefore, the change degree of the organism inflammation can be known by detecting the concentration change of substances such as NO, TNF-alpha, IL-6 and the like in the organism.

Polysaccharides are widely found in plants, fungi, microorganisms and animals, with plants being one of the most abundant sources of polysaccharides. Among them, acidic polysaccharides (containing uronic acid or sulfate) have been receiving increasing attention due to their low toxicity and potential biological activity, and their biological functions vary with the chemical structures such as molecular weight, uronic acid content, branching degree, glycosidic bond, etc. Thus, the structural features of acidic polysaccharides are the basis for their biological activity. Many studies have shown that acidic polysaccharides can be an ideal natural anti-inflammatory drug, and can resist external stimuli by inhibiting secretion of inflammatory cytokines, which is a hot spot of research in recent years.

Ampelopsis Grossedentata (AG), also known as Ampelopsis grossedentata, is widely distributed in southwest China, and leaves thereof have been used as traditional health tea and Chinese herbal medicines for thousands of years. AG is known in the traditional chinese medicine to have many beneficial effects including clearing heat and detoxicating, diminishing inflammation and promoting urination, promoting blood circulation and eliminating blood stasis. AG has long been used as a medicine for treating colds, stings, bruises, hepatitis and sore throat. AG is known to contain high levels of polyphenols, flavonoids, polysaccharides, alkaloids and fatty acids from modern scientific research. Modern pharmacological studies show that AG aqueous extract has anti-inflammatory effect, and obviously, the AG aqueous extract contains polysaccharide which is completely ignored by the previous studies, and the connection between the structural characteristics and the anti-inflammatory activity is still unknown.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the primary purpose of the invention is to provide vine tea acidic polysaccharide AGP-3a.

The invention also aims to provide an extraction and separation method of the vine tea acidic polysaccharide AGP-3 a.

It is still another object of the present invention to provide an application of the above vine tea acidic polysaccharide AGP-3a in preparing anti-inflammatory cosmetics.

The aim of the invention is achieved by the following technical scheme:

the monosaccharide molecular composition chain of the vine tea acid polysaccharide AGP-3a is shown as the following formula (I):

the weight average molecular weight of the vine tea acid polysaccharide AGP-3a is 278kDa;

the vine tea acid polysaccharide AGP-3a consists of mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara) monosaccharides, wherein the molar ratio of the monosaccharides is 23.31:18.08:10.06:11.36:26.84:10.35.

Structural analysis of the vine tea acid polysaccharide AGP-3a shows that 1,6-LINKED GALP and 1,3,6-LINKED MANP are the main skeletons thereof.

The extraction and separation method of the vine tea acidic polysaccharide AGP-3a comprises the following operation steps:

(1) Taking the dried commercial vine tea water extract, using water for water bath at 85 ℃ until the water extract is completely dissolved, and adding absolute ethyl alcohol when the water extract is hot until the volume percentage concentration of the ethyl alcohol in the solution reaches 60-65%; cooling the solution at 4deg.C, filtering to obtain precipitate, and washing with anhydrous ethanol to obtain Ampelopsis grossedentata crude polysaccharide; removing protein from the vine tea crude polysaccharide by a Sevag method, and removing pigment by adopting active carbon to obtain pure vine tea crude polysaccharide;

(2) Dissolving the pure vine tea crude polysaccharide obtained in the step (1) into ultrapure water, passing through a DEAE-sepharose CL-6B column, performing gradient elution by adopting 0-0.3mol/LNaCl solution, collecting 10mL of the pure vine tea crude polysaccharide from each tube at the flow rate of 2.2mL/min, and combining and collecting 140-175 Guan Xituo th peak solution;

(3) Combining the 140 th to 175 th Guan Xituo th peak solutions collected in the step (2), dialyzing for 48 hours by using a 3.5kDa dialysis bag, concentrating under reduced pressure, and freeze-drying to obtain vine tea polysaccharide;

(4) Dissolving the vine tea polysaccharide obtained in the step (3) in ultrapure water, passing through a 0.45 mu m filter membrane, passing through a sephadex-G75 chromatographic column, eluting with ultrapure water at a flow rate of 0.3mL/min, collecting 3mL of the vine tea polysaccharide per tube, combining and collecting elution peak parts in the 9 th to 30 th tubes, concentrating under reduced pressure, and freeze-drying to obtain vine tea acidic polysaccharide AGP-3a with uniform molecular weight.

The protein of the vine tea crude polysaccharide is removed by a Sevag method in the step (1), and the method specifically comprises the following steps: re-dissolving Ampelopsis grossedentata crude polysaccharide with clear water, concentrating under reduced pressure until ethanol is removed, and obtaining Ampelopsis grossedentata crude polysaccharide liquid; the preparation method comprises the following steps of: adding Sevag reagent into the vine tea crude polysaccharide liquid according to the volume ratio of Sevag reagent of 4:1, wherein the Sevag reagent is mixed liquid of chloroform and n-butanol according to the volume ratio of 4:1, magnetically stirring for 25min, and centrifuging at the temperature of 4 ℃ for 15min at the rotating speed of 10000r/min to obtain supernatant; repeating the operation of removing protein by Sevag method for multiple times, and combining the supernatant to obtain crude vine tea polysaccharide with protein removed;

the method for removing pigment by using activated carbon specifically comprises the following steps: adding active carbon into the crude vine tea polysaccharide with protein removed, magnetically stirring in water bath at 50deg.C for 10min to remove pigment, filtering to obtain filtrate, concentrating under reduced pressure, and freeze drying to obtain pure crude vine tea polysaccharide.

The application of the vine tea acidic polysaccharide AGP-3a in preparing anti-inflammatory cosmetics.

The principle of the invention is as follows:

The vine tea polysaccharide is present in vine tea material and may be concentrated in dried vine tea water extract via water extraction. Dissolving the dried vine tea water extract with water, precipitating the vine tea water extract in a water extraction and alcohol precipitation mode, removing protein and active carbon by a Sevag method, removing pigment, finally using a DEAE-sepharose CL-6B column and a sephadex-G75 chromatographic column with molecular sieve effect, gradually separating and purifying the vine tea water extract by combining gradient elution, and finally obtaining vine tea acidic polysaccharide AGP-3a with uniform molecular weight by collecting eluent in a certain range.

In the course of studying the anti-inflammatory activity of tendrilleaf polysaccharide, since inflammatory response is a beneficial self-protecting defensive response of the body to stimulation, macrophages in the innate immune system release cytokines associated with inflammation such as Nitric Oxide (NO), tumor necrosis factor (TNF- α), interleukin-6 (IL-6) and the like after being stimulated during the course of inflammation. Therefore, the vine tea polysaccharide can be added into the macrophage culture medium, and whether the vine tea polysaccharide has anti-inflammatory activity can be known by comparing and detecting the concentration changes of substances such as NO, TNF-alpha, IL-6 and the like in the macrophage after the macrophage is stimulated.

Compared with the prior art, the invention has the following advantages and effects:

(1) According to the extraction and separation process of the invention, an acidic polysaccharide AGP-3a with uniform molecular weight and a number average molecular weight of 278kDa can be prepared.

(2) The acidic polysaccharide AGP-3a obtained by the invention has excellent anti-inflammatory effect, and can be used for products such as cosmetics, medicines and the like.

(3) The invention further analyzes the composition and structure of the active ingredients in the vine tea, and provides a theoretical basis for deep understanding of the material basis of the physiological activity of the vine tea.

Drawings

FIG. 1 is an elution profile of DEAE-sepharose CL-6B for separating Ampelopsis grossedentata crude polysaccharide.

FIG. 2 is an elution profile of the Sephadex G75 column purification of the Ampelopsis grossedentata acidic polysaccharide AGP-3a component.

FIG. 3 is a compositional analysis of vine tea acidic polysaccharide AGP-3a monosaccharide.

FIG. 4 is a scan of vine tea acid polysaccharide AGP-3a in the range 4000-400cm-1 using FT-IR spectrometer.

FIG. 5 is a 1H NMR chart of vine tea acid polysaccharide AGP-3 a.

FIG. 6 is a 13C NMR chart of vine tea acid polysaccharide AGP-3 a.

FIG. 7 is a HSQC chart of the vine tea acid polysaccharide AGP-3 a.

FIG. 8 is a COSY pattern of vine tea acid polysaccharide AGP-3 a.

FIG. 9 is a HMBC pattern of the vine tea acid polysaccharide AGP-3 a.

FIG. 10 is NOSEY spectral chart of vine tea acid polysaccharide AGP-3a

FIG. 11 is a plan view of the acidic polysaccharide AGP-3a of Ampelopsis grossedentata in aqueous solution.

FIG. 12 is a three-dimensional image of the vine tea polysaccharide AGP-3a polysaccharide.

FIG. 13 is the maximum absorption wavelength of vine tea acidic polysaccharide AGP-3a Congo red polysaccharide complex in NaOH solutions of different concentrations.

FIG. 14 is an SEM analysis of the vine tea acid polysaccharide AGP-3 a.

FIG. 15 is the effect of the vine tea acid polysaccharide AGP-3a on macrophage RAW264.7 cell activity.

FIG. 16 is the effect of the vine tea acid polysaccharide AGP-3a on NO secretion by macrophage RAW264.7 cells.

FIG. 17 is the effect of the vine tea acid polysaccharide AGP-3a on secretion of TNF-alpha by macrophage RAW 264.7.

FIG. 18 is the effect of the vine tea acid polysaccharide AGP-3a on IL-6 secretion by macrophage RAW264.7 cells.

Detailed Description

The present invention is further illustrated below in conjunction with specific examples, but should not be construed as limiting the invention.

The data processing method in the following embodiments is: all experiments were performed in triplicate; adopting SPSS 19.0 software package to process data; the significance level was taken as 0.05 and the data were expressed as mean ± standard deviation.

Example 1: preparation, separation and purification of vine tea acidic polysaccharide AGP-3a

1. Taking dried vine tea water extract (20 g), using 200ml of purified water to dissolve completely in a water bath at 85 ℃, and adding 3 times of absolute ethyl alcohol in volume while the vine tea water extract is hot until the volume percentage concentration of the ethyl alcohol in the solution is 60-65%;

2. Cooling the solution at 4deg.C, filtering to obtain precipitate, and washing to obtain Ampelopsis grossedentata crude polysaccharide;

3. Re-dissolving the vine tea crude polysaccharide obtained in the step 2 by using clear water to 100ml, and concentrating under reduced pressure until ethanol is removed to obtain vine tea crude polysaccharide solution; the preparation method comprises the following steps of: adding Sevag reagent into the Ampelopsis grossedentata crude polysaccharide solution according to the volume ratio of Sevag reagent of 4:1, magnetically stirring for 25min, centrifuging at 4 ℃ for 15min (the rotating speed is 10000 r/min), and combining the supernatant (the protein removal process by Sevag method is repeated for 5 times) to obtain the Ampelopsis grossedentata crude polysaccharide with protein removed;

4. adding activated carbon (0.4 g) into the Ampelopsis grossedentata crude polysaccharide with proteins removed, magnetically stirring in water bath at 50deg.C for 10min to remove pigment, filtering to obtain filtrate, concentrating under reduced pressure, and freeze drying to obtain pure Ampelopsis grossedentata crude polysaccharide;

5. Dissolving pure Ampelopsis grossedentata crude polysaccharide (150 mg) obtained in the step 4 in ultrapure water (10 mL), centrifuging at 10000r/min at 4 ℃ for 15min, filtering supernatant with a 0.45 μm filter membrane, passing through DEAE-agarose gel CL-6B, gradient eluting with 0-0.3mol/L NaCl solution, and collecting 10mL per tube at a flow rate of 2.2 mL/min. The polysaccharide component was detected by phenol-sulfuric acid method, and the elution curve was drawn with the number of tubes for collecting the eluate as abscissa and the absorbance of the sample at 490nm as ordinate, as shown in FIG. 1.

6. According to the elution curve of FIG. 1, the 140 th to 175 th Guan Xituo th peak solutions collected are combined, dialyzed for 48 hours by a dialysis bag (3.5 kDa), concentrated under reduced pressure, and freeze-dried to obtain vine tea polysaccharide AGP-3 with higher purity (i.e. higher uniformity of molecular weight).

7. Dissolving 50mg of vine tea polysaccharide AGP-3 obtained in the step 6 in ultrapure water (5 mL), passing through a 0.45 mu m filter membrane, passing through a sephadex-G75 chromatographic column, eluting with ultrapure water at a flow rate of 0.3mL/min, collecting 3mL of the mixture per tube, detecting polysaccharide components by using a phenol-sulfuric acid method, and drawing an elution curve by taking the number of tubes for collecting the eluent as an abscissa and taking absorbance of the sample at 490nm as an ordinate, wherein the elution curve is shown in figure 2. According to the elution profile of FIG. 2, the elution peak portions in the 9 th to 30 th tubes were collected together, concentrated under reduced pressure, and freeze-dried to obtain vine tea acidic polysaccharide AGP-3a (37.8 mg) having a uniform molecular weight.

As can be seen from FIG. 1, after the crude vine tea polysaccharide after deproteinization and pigment removal is subjected to gradient elution (0-0.3 mol/L NaCl) through DEAE-agarose gel CL-6B, the crude vine tea polysaccharide can be separated into 4 vine tea polysaccharide components with relatively uniform molecular weights, such as AGP-0, AGP-1, AGP-2, AGP-3 and the like.

As can be seen from FIG. 2, the collected and lyophilized AGP-3 was further purified by a Sephadex-G75 column, eluted with ultra pure water at a flow rate of 0.3mL/min, collected 3mL per tube, and assayed for sugar content by the phenol sulfuric acid method, and the eluted peak fractions from the 9 th to 30 th tubes were collected (FIG. 2), concentrated under reduced pressure, and freeze-dried to obtain the more pure (more uniform molecular weight) vine tea extract polysaccharide AGP-3a. The following analysis was performed on vine tea extract polysaccharide AGP-3 a:

(1) Molecular weight

Molecular weight determination of AGP-2a (10 mg/mL) was performed using an HPLC system, equilibrated with Ultranhydrogel Linear chromatography column (30 cm. Times.7.8 mm) with 25mmol/L NaH2PO4-25mmol/LNa2HPO4 (pH 6.7,0.05% NaN 3) as eluent (flow = 0.8 mL/min). The eluate was monitored using a differential refractive detector. The column was calibrated by injection of dextran standards (10 μl) of known molecular weights (1.0, 5.0, 12.0, 25.0, 50.0, 150.0 and 670.0 kDa). The molecular weight of AGP-3a is about 278kDa, which can be obtained from the standard curve equation for different dextran standards, the analytical module carried by the detection instrument itself and the retention time of AGP-3 a.

(2) Analysis of monosaccharide composition

Comparing the chromatograms of the mixed standard monosaccharide and polysaccharide fractions, it can be seen that the AGP-3a polysaccharide fraction, consisting of Man, glcA, galA, glc, gal and Ara, is a typical acidic polysaccharide (FIG. 3). The molar ratio of AGP-3a was 23.31:18.08:10.06:11.36:26.84:10.35, indicating that Man and Gal may be the main backbones of AGP-3 a.

(3) Methylation analysis

The peak of AGP-3a methylated sugar was identified using retention time and mass spectrometry of the methylated sugar alcohol, from which 6 derivatives of Terminal-Glcp,1,5-LINKED ARAF,1,6-LINKED GALP,1,2-LINKED GLCP,1,4-LINKED GALP,1,3,6-LINKED MANP, etc. were found. The ratio of each glycosidic bond was obtained from the peak area (see Table 1) and the results were substantially consistent with the monosaccharide composition analysis. The maximum ratio of 1,6-LINKED GALP and 1,3,6-LINKED MANP indicates that the two glycosidic bonds form the skeleton of AGP-3 a.

Methylation analysis of Table 1 AGP-3a

(4) Infrared spectroscopic analysis

The FT-IR spectrum of AGP-3a is shown in FIG. 4, and has more absorption peaks in the range of 400-4000 cm-1. Wherein, two absorption peaks are respectively related to the stretching vibration of O-H and the asymmetric stretching vibration of C-H at 3412.42cm < -1 > and 2935.70cm < -1 >. Furthermore, the peak at 1614.44cm-1 indicates the presence of uronic acid, and the peak at 1417.32cm-1 is the flexural vibration of C-H. Meanwhile, the C-O-H stretching vibration and the C-O-C glycoside band vibration showed strong absorption peaks at 1145.72cm-1, 1080.15cm-1 and 1020.36cm-1, which confirm the presence of the pyranose ring. FT-IR results indicated that AGP-3a has a typical polysaccharide absorption peak.

(5) Nuclear magnetic resonance analysis

Based on monosaccharide composition and methylation analysis results, chemical shifts of all sugar residues were attributed to the combination of 1D (1 h,13 c) and 2D (COSY, HSQC, HMBC) NMR (table 2), and the relationship between the residues was obtained, finally obtaining the polysaccharide chain structure.

TABLE 2 1H and 13C NMR chemical shift analysis of AGP-3a

AGP-3a showed 6 ectopic proton signals at δ 5.31,5.13,5.00,4.56,4.44,4.36 according to the cross peaks of 1H NMR (FIG. 5), 13C NMR (FIG. 6) and HSQC (FIG. 7). The corresponding ectopic carbon signals were delta 98.28,100.86,107.43,104.22,102.57,101.34ppm (labeled a, b, c, d, e, f, respectively), indicating that AGP-3a all had alpha-and beta configurations, consistent with the presence of weak peaks in FT-IR between 800cm-1 and 900 cm-1. Proton signal of δ1.90-2.00ppm in 1H NMR and carbon signal of δ20.22 in 13C NMR indicate that AGP-3a has acetyl. Carbon signals of delta 52.66ppm and delta 174.16ppm indicate the presence of methyl and uronic acid, respectively, in the polysaccharide.

For residue a in AGP-3a, strong ectopic proton and ectopic carbon signals were found at δ5.31ppm and δ98.28ppm, indicating that residue A is the alpha-isomerised carbon in pyranose. The presence of the cross-peaks of δ5.31/4.07,4.07/3.48,3.48/3.87,3.87/3.64 in the COSY (FIG. 8) spectrum indicates that the H2-H5 signal for residue a is at δ4.07,3.48,3.87,3.64ppm the corresponding C2-C5 signal obtained using the HSQC spectrum is δ77.33,71.53,73.14,72.5ppm and the top field shift of C2 indicates the presence of the (1.fwdarw.2) bond for residue a. Meanwhile, δ3.64/174.88 and δ3.74/174.88 in HMBC spectra indicate that the hydroxyl group of uronic acid is esterified, and thus residue a is identified as → 2) - α -D-GlcpA-6-OMe- (1 → (Patra et al, 2013). The remaining residues were further analyzed by similar rules using COSY and HSQC, and the chemical shift was completely assigned to all protons and carbon signals, and the results are shown in table 2.

The HMBC (FIG. 9) diagram of AGP-3a shows that δ3.64/174.88 shows that the hydroxyl groups of the uronic acid are esterified and δ3.74/174.88 shows that the uronic acid is located in residues a and e. The sugar chain structure of AGP-3a cannot be estimated by HMBC without crossing peaks of the carbon signal displaced from the top field in the region of the ectopic proton signal (. Delta.4.36-5.31). NOESY spectroscopy is another detection method that can determine the linkage of sugar residues in polysaccharides, reflecting proton signals within the residues and proton signal cross peaks between the residues (Guo, et al, 2018). The NOSEY spectrum of AGP-3a is shown in FIG. 10, where H1 (δ5.31) at residue a has a cross-peak with H6 (δ3.31) at residue f and H4 (δ3.68) at residue e, respectively, demonstrating the presence of a-f and a-e linkages. H1 (δ5.13) at residue b has a cross peak with H2 (δ4.07) at residue a, H6 (δ3.31) at residue f, H5 (δ3.82) at residue c and H6 (δ3.71) itself, respectively, demonstrating the presence of b-a, b-b, b-c and b-f linkages. H1 (δ5.31) at residue c has a cross peak with H6 (δ3.71) at residue b and H3 (δ3.94) at residue f, respectively, demonstrating the presence of c-b and c-f linkages. H1 (δ4.56) at residue d has a cross peak with H2 (δ4.07) at residue a and H6 (δ3.71) at residue b, respectively, demonstrating the presence of d-a and d-b linkages. Similarly, delta 4.44/3.94, delta 4.36/3.31, delta 4.36/4.07 and delta 4.36/3.3.71 demonstrate the presence of e-f, f-a, f-b and f-f links, respectively.

In summary, the constituent chains of the monosaccharide molecules of AGP-3a are shown in formula (I):

(6) AFM analysis

The experiments were performed using Atomic Force Microscopy (AFM) to observe the ultrastructural and surface morphology of AGP-3 a. As a result, it was found that AGP-2a had a height of about 1.5nm, and that the planar image showed an AGP-3a distribution and a uniform concentration of the distribution (FIG. 11).

Three-dimensional images of AGP-3a polysaccharide (FIG. 12) all showed an uneven surface morphology, consisting of a number of peak-like protruding structures of varying height, arranged in an irregular protruding structure, indicating that AGP-3a has a branched structure.

(7) Congo red analysis

Typically, upon binding of the triple helix polysaccharide to congo red, the maximum absorption wavelength (λmax) of the congo red-polysaccharide complex will undergo a red shift, with a tendency to rise and fall. Thus, the Congo red method can be used to determine the triple helix junction of AGP-3 a. As shown in FIG. 13, the maximum absorption wavelength of AGP-3a decreases with increasing NaOH concentration, indicating that AGP-3a does not have a triple helix structure.

(8) SEM analysis

The spatial structure of polysaccharides is generally more complex than nucleotides and proteins. The SEM results of AGP-3a are shown in FIG. 14. The appearance features of the product are compact lamellar structure, and the product is very smooth on the surface and almost has no cracks.

Example 2: anti-inflammatory Activity test of Ampelopsis grossedentata extract polysaccharide AGP-3a

1. Ampelopsis grossedentata extract polysaccharide AGP-3a can improve macrophage activity, and has no cytotoxicity.

RAW264.7 macrophages (supplied by the national academy of sciences cell bank, shanghai, china) were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin, and the cell culture was performed in an incubator with a humidified atmosphere of 5% CO2 at 37 ℃. RAW264.7 cells were then cultured in 96-well plates at a density of 1X 105cells/mL for 24 hours. The medium was removed, and fresh medium containing AGP-3a (0.2-1.6 mg/mL) obtained in example 1 at various concentrations was added for culturing for 24 hours, and the cell viability was measured by the CCK-8 method.

As can be seen from FIG. 15, RAW264.7 cell activity was enhanced with the addition of 200, 400, 800 and 1600 μg/mLAGP-3a concentrations (NC is a control without AGP-3a addition), indicating that AGP-3a had no inhibitory effect on RAW264.7 cell viability and no cytotoxicity.

2. AGP-3a in vitro anti-inflammatory Activity

RAW264.7 macrophages were seeded in 96-well plates. After culturing at a density of 1X 105cells/mL for 24 hours, fresh medium of AGP-3a (0.2-1.6 mg/mL) obtained in example 1 was used at different concentrations for 2 hours, respectively. DMEM medium (blank medium without LPS) containing LPS (1. Mu.g/mL) was then added. After 24h incubation, IL-6 and TNF- α levels in the supernatants were detected using mouse IL-6 and TNF- α ELISA kits, respectively, while NO content in the supernatants was determined using NO detection kit.

LPS is the main component of the cell wall of gram-negative bacteria, and can bind to the TLR 4 receptor of macrophages to induce inflammation. Thus, LPS was selected as the stimulator to activate macrophages at the cellular level of RAW264.7 cells, mimicking inflammatory disease without cytotoxicity. After the addition of LPS to the medium, macrophages react to LPS, synthesize and release inflammatory mediators, such as NO, and produce pro-inflammatory cytokines, such as TNF- α and IL-6. The elevation of the pro-inflammatory cytokines TNF- α and IL-6 is considered a typical marker of acute inflammatory responses.

As can be seen from fig. 16-18, the expression levels of NO, TNF- α and IL-6 were all significantly increased (p < 0.05) after LPS activation of RAW264.7 cells compared to the blank control (NC), indicating that inflammation was stimulated.

As can be seen from FIG. 16, AGP-3a showed an inhibitory effect on the production of NO by LPS-activated cells after addition of AGP-3a to the medium, and had a remarkable inhibitory effect (p < 0.05) at a concentration of more than 200. Mu.g/mL, and the higher the concentration, the better the inhibitory effect. When the concentration reaches 1600 mug/mL, the NO expression level is reduced by 65.87% after the LPS activated cell is treated by AGP-3 a.

From FIGS. 17 and 18, it can be seen that AGP-3a showed an inhibitory effect on TNF- α and IL-6 production by LPS-activated cells, a significant inhibitory effect (p < 0.05) at all concentrations, and a concentration dependence. When the concentration reaches 1600 mug/mL, the TNF-alpha expression level is reduced by 32.63% after the LPS is activated by AGP-3a treatment, and the IL-6 expression level is reduced by 38.17%, thus the result shows that the AGP-3a shows better anti-inflammatory activity.

From the above results, AGP-3a has a remarkable anti-inflammatory activity in vitro.

Example 3 preparation of anti-inflammatory cosmetics in different dosage forms

1. The tenuifolia polysaccharide toner for anti-inflammatory can be prepared according to a conventional method by mixing: 2.5% by weight of the vine tea polysaccharide AGP-3a obtained in example 1, 4.5% by weight of 1, 3-butanediol, 2.3% by weight of oleyl alcohol, 3.0% by weight of ethanol, 2.4% by weight of polysorbate 20, 3.1% by weight of benzophenone-9, 0.9% by weight of carbopol, 3.5% by weight of glycerol, trace amounts of perfume, trace amounts of preservative, and the balance purified water.

2. The vine tea polysaccharide nutrition cream for anti-inflammatory is prepared according to the conventional method by mixing: 5.0% by weight of the vine tea polysaccharide AGP-3a obtained in example 1, 3.5% by weight of glycerol, 3.7% by weight of vaseline, 2.4% by weight of triethanolamine, 5.0% by weight of liquid paraffin, 3.3% by weight of squalane, 3.0% by weight of beeswax, 5.0% by weight of tocopheryl acetate, 3.0% by weight of polysorbate 60, 1.2% by weight of carbopol, 3.1% by weight of sorbitan sesquioleate, trace amounts of perfume, trace amounts of preservative, and the balance purified water.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (4)

1. Use of vine tea acidic polysaccharide AGP-3a with anti-inflammatory activity in the preparation of anti-inflammatory cosmetics, characterized in that: the monosaccharide molecular composition chain of the vine tea acidic polysaccharide AGP-3a is shown as the following formula (I):

the weight average molecular weight of the vine tea acid polysaccharide AGP-3a is 278kDa;

the vine tea acid polysaccharide AGP-3a consists of mannose (Man), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara) monosaccharides, wherein the molar ratio of the monosaccharides is 23.31:18.08:10.06:11.36:26.84:10.35.

2. Use according to claim 1, characterized in that: structural analysis of the vine tea acid polysaccharide AGP-3a shows that 1,6-LINKED GALP and 1,3,6-LINKED MANP are the main skeletons thereof.

3. Use according to claim 1, characterized in that: the vine tea acid polysaccharide AGP-3a is prepared according to the following operation steps:

(1) Taking the dried commercial vine tea water extract, using water for water bath at 85 ℃ until the water extract is completely dissolved, and adding absolute ethyl alcohol when the water extract is hot until the volume percentage concentration of the ethyl alcohol in the solution reaches 60-65%; cooling the solution at 4deg.C, filtering to obtain precipitate, and washing with anhydrous ethanol to obtain Ampelopsis grossedentata crude polysaccharide; removing protein from the vine tea crude polysaccharide by a Sevag method, and removing pigment by adopting active carbon to obtain pure vine tea crude polysaccharide;

(2) Dissolving the pure vine tea crude polysaccharide obtained in the step (1) into ultrapure water, passing through a DEAE-sepharose CL-6B column, performing gradient elution by adopting 0-0.3mol/LNaCl solution, collecting 10mL of the pure vine tea crude polysaccharide from each tube at the flow rate of 2.2mL/min, and combining and collecting 140-175 Guan Xituo th peak solution;

(3) Combining the 140 th to 175 th Guan Xituo th peak solutions collected in the step (2), dialyzing for 48 hours by using a 3.5kDa dialysis bag, concentrating under reduced pressure, and freeze-drying to obtain vine tea polysaccharide;

(4) Dissolving the vine tea polysaccharide obtained in the step (3) in ultrapure water, passing through a 0.45 mu m filter membrane, passing through a sephadex-G75 chromatographic column, eluting with ultrapure water at a flow rate of 0.3mL/min, collecting 3mL of the vine tea polysaccharide per tube, combining and collecting elution peak parts in the 9 th to 30 th tubes, concentrating under reduced pressure, and freeze-drying to obtain vine tea acidic polysaccharide AGP-3a with uniform molecular weight.

4. Use according to claim 3, characterized in that:

the protein of the vine tea crude polysaccharide is removed by a Sevag method in the step (1), and the method specifically comprises the following steps: re-dissolving Ampelopsis grossedentata crude polysaccharide with clear water, concentrating under reduced pressure until ethanol is removed, and obtaining Ampelopsis grossedentata crude polysaccharide liquid; the preparation method comprises the following steps of: adding Sevag reagent into the vine tea crude polysaccharide liquid according to the volume ratio of Sevag reagent of 4:1, wherein the Sevag reagent is mixed liquid of chloroform and n-butanol according to the volume ratio of 4:1, magnetically stirring for 25min, and centrifuging at the temperature of 4 ℃ for 15min at the rotating speed of 10000r/min to obtain supernatant; repeating the operation of removing protein by Sevag method for multiple times, and combining the supernatant to obtain crude vine tea polysaccharide with protein removed;

the method for removing pigment by using activated carbon specifically comprises the following steps: adding active carbon into the crude vine tea polysaccharide with protein removed, magnetically stirring in water bath at 50deg.C for 10min to remove pigment, filtering to obtain filtrate, concentrating under reduced pressure, and freeze drying to obtain pure crude vine tea polysaccharide.