JP7414303B2 - Stir-fried homogeneous polysaccharide and its preparation method and application - Google Patents

Description

TECHNICAL FIELD The present invention relates to the technical field of pharmaceutical science, and specifically relates to a homogeneous polysaccharide and its preparation method and application.

Acanthopanax senticosus (Rupr. Et Maxim) Harms is a dried root and rhizome or stem derived from Acanthopanax senticosus (Rupr. Et Maxim) Harms, a plant in the genus Acanthopanax of the family Acanthopanax, which is grown in the coniferous forest zone of the Russian Far East, northeastern China, It is widely distributed in Hebei province, Shanxi province, Japan, and the northern part of North Korea. It is also known as the "Wu Jia Jian", the "Stab Stick", and the "Old Tiger Lie Zi". In Traditional Chinese Medicine, the use of Chigoka as a drug has a long history, and it has been described in successive Chinese herbal works. Sue Hongkei of the Southern Dynasty wrote in a book called ``Famous Doctor Bessroku'' that the ``five leaves'' of sashigoka have the effect of ``conditioning the inside, benefiting the spirit, strengthening the muscles, and strengthening the will.'' It points out that. Li Shizhen of the Ming dynasty said that zhujiujia is ``a superior product in the herbal medicine of Shennong,'' and that it ``conditions the inside, benefits the Qi, strengthens the muscles, strengthens the will, and when taken for a long time, it makes you feel light and resists aging.'' '' is shown in the ``Honso Tsuname''. Zing Wuji has medicinal properties of warmth, is spicy and slightly bitter, and acts on the spleen, kidney, and heart meridians, increasing Qi, normalizing the function of the spleen and stomach, improving the function of the kidney, and improving the mental health. It has a stabilizing effect, and is mainly used to treat spleen, kidney, yang deficiency, physical weakness, loss of appetite, pain in the lower back and knees, and insomnia. (Geong-ji Ban, Jin-ha, Wen-ok Choi et al., Advances in research on the chemical components and pharmacological activities of Chingoka [J]. Food Industry Science and Technology, 2019, 40(23): 353-360) Chingoka can regulate immune function. It has various pharmacological effects such as anti-cancer, liver protection, anti-aging, antioxidant, anti-inflammation, lowering blood pressure, and anti-stress, and its active ingredients include syringins, flavonoids, lignans, and polysaccharides. However, these are experimental results for crude polysaccharides that have low reproducibility and cannot be used as standard products, and their value for industrialization is limited. The purpose of the present invention is to isolate and purify the crude polysaccharide of Sashigoka, analyze its structure, and evaluate its biological activity based on previous research, and to obtain a homogeneous polysaccharide of Sashigoka with good biological activity. , not only has very important scientific significance, but also can lay the foundation for the industrialization of polysaccharides.

The technical problem to be solved by the present invention is to grade polysaccharides according to their molecular weights, then further separate and purify them and structurally characterize them. The purpose of the present invention is to provide a homogeneous polysaccharide containing sterilized polysaccharides, and to perform preliminary research on the antioxidant activity and anti-skin aging activity of the homogeneous polysaccharide obtained.

The object of the present invention has a molecular weight of 6.83×10 ⁵ Da and is composed of fucose, arabinose, galactose, glucose , and xylose, and the mole percentage of each monosaccharide is 16.42%, 32.27%, and 40%, respectively. .38%, 7.21% and 3.72%.

Another object of the present invention is to provide a method for preparing the homogeneous polysaccharide, which comprises the following steps:
1) After crushing the dried Sashigoka drink pieces and passing them through a sieve, add 5 to 8 times the amount of water to the medicinal powder, and extract at a temperature of 80 to 100°C for 2 hours each time, 3 times in total to prepare the extract. combining, centrifuging to collect the supernatant, and concentrating the supernatant to obtain a concentrate;
2) After cooling the concentrate, add α-amylase to a weight of 0.1 to 0.4%, adjust the pH value to 7.0, and add iodine-iodine in a water bath at 60°C. Exposing the solution to a potassium reagent to perform enzymatic decomposition until the solution no longer changes color, rapidly raising the temperature to 100°C and holding for 5 minutes to inactivate the enzyme, and collecting the supernatant by centrifugation;
3) Mix the supernatant liquid collected in step 2) and Sevage reagent at a volume ratio of 1:1, shake vigorously for 30 min, let stand for 12 h, collect the upper layer polysaccharide solution, and mix with the upper layer polysaccharide solution and Sevage reagent. mixing the reagents in a 1:1 volume ratio and repeating the above operation until the characteristic absorption peak of the protein disappears in the UV scan;
4) After concentrating the upper polysaccharide solution finally collected in step 3), add 4 to 6 times the volume of absolute ethanol, let it stand at 4°C for 48 hours to precipitate it, and then centrifuge it. collecting the precipitate, adding absolute ethanol to the precipitate, repeating the above operation three times, and then freeze-drying it to obtain a crude polysaccharide powder of Sashigoka;
5) After completely dissolving the crude polysaccharide powder of Sashigoka obtained in step 4) in distilled water, it was separated using a DEAE Fast Flow ion chromatography column, and the elution conditions were a flow rate of 2.5 mL/min. , pure water, 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 1 mol/L sodium chloride solution, followed by follow-up detection using the sulfuric acid-phenol method. performing and collecting the eluate;
6) After concentrating the eluate collected in step 5), it was reseparated using a Sephadex G-200 dextran gel column, eluted with distilled water at a flow rate of 0.5 mL/min, and detected using the phenol-sulfuric acid method. collecting a main peak portion of the elution curve;
7) After concentrating the solution in the main peak part of the elution curve collected in step 6), it is desalted by dialysis for 2 days using a dialysis bag with a cut-off molecular weight of 3500 Da, and finally the solution in the dialysis bag is concentrated. freeze-drying to obtain a homogeneous polysaccharide powder.

The present invention provides a method for preparing sashigoka homogeneous polysaccharide, which includes the following steps:
1) Grind the dried sashigoka drink and pass it through a 100 mesh sieve.The sieved medicinal powder is mixed with 5 to 8 times its weight of water at a temperature of 80 to 100°C for 2 hours each time, 3 times in total. extracting, combining the extracts, centrifuging at 3000 rpm/min for 20 min to collect the supernatant, and concentrating the supernatant to one-fifth of the original volume by rotary evaporation to obtain a concentrate;
2) After cooling the concentrate, add α-amylase to a concentration of 0.1 to 0.4% by weight, adjust the pH value to 7.0, and add iodine to iodine in a water bath at 60°C for 4 h. Enzyme digestion was carried out (until the solution no longer changed color by exposure to a potassium chloride reagent), then the temperature was rapidly raised to 100°C and held for 5 minutes to inactivate the enzyme, followed by centrifugation at 3000 rpm/min for 10 minutes. collecting the supernatant;
3) The supernatant collected in step 2) and Sevage reagent (chloroform: n-butanol mixed at a volume ratio of 4:1) were mixed at a volume ratio of 1:1, shaken vigorously for 30 min, and then left to stand still for 12 h. collecting the upper layer polysaccharide solution, mixing the upper layer polysaccharide solution and Sevage reagent in a volume ratio of 1:1, and repeating the above operation until the characteristic absorption peak of the protein disappears in the UV scan;
4) After concentrating the upper layer polysaccharide solution finally collected in step 3), add 4 to 6 times the volume of absolute ethanol, let it stand at 4°C for 48 hours to precipitate it, and then turn the solution at 3000 rpm/min. Collect the precipitate by centrifugation for 10 min, add absolute ethanol to the precipitate, repeat the above operation three times, freeze-dry the final precipitate, and prepare the crude polysaccharide of sashigoka. obtaining a powder;
5) After completely dissolving the crude polysaccharide powder of Sashigoka obtained in step 4) in distilled water, it was separated using a DEAE Fast Flow ion chromatography column, and the elution conditions were a flow rate of 2.5 mL/min. , pure water, 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 1 mol/L sodium chloride solution, and then the eluate was collected using a fully automatic collector. Collect 30 tubes with a gradient, elute each gradient solution with 3 column volumes, 5.0 mL per tube, and perform septum tube follow-up detection using the sulfuric acid-phenol method. and,
6) After concentrating the eluate collected in step 5), it was reseparated using a Sephadex G-200 dextran gel column, eluted with distilled water at a flow rate of 0.5 mL/min, and collected per tube using a fully automatic collector. collecting the main peak portion of the elution curve after detecting with phenol-sulfuric acid;
7) After concentrating the solution in the main peak part of the elution curve collected in step 6), it is desalted by dialysis for 2 days using a dialysis bag with a cut-off molecular weight of 3500 Da, and finally the solution in the dialysis bag is concentrated. freeze-drying to obtain a homogeneous polysaccharide powder.

Further, in the Sevage reagent, the volume ratio of chloroform:n-butanol is 4:1.

In the present invention, when extracted by a water extraction ethanol precipitation method and further separated and purified using an anion exchange DEAE Fast Floe chromatography column and a Superdex-200 gel chromatography column, the molecular weight is 6.83×10 ⁵ Da. We found that a homogeneous polysaccharide consisting of fucose , arabinose, galactose, glucose , and xylose can be obtained.

The test results show that the homogeneous polysaccharide powder prepared according to the present invention has various effects such as antioxidant and anti-skin aging, and can be used in the preparation of anti-aging cosmetics and skin treatments. Proven.

The present invention further relates to skin care cosmetics comprising the above-mentioned polysaccharides having anti-skin aging effects and excipients used in the cosmetic field.

The present invention further relates to a dermatological therapeutic agent comprising a pentagonal homogeneous polysaccharide having an anti-skin aging effect as described above, and a medically acceptable carrier.

The stinging homogeneous polysaccharide provided by the present invention can be used in particular for the preparation of skin care cosmetics such as creams, emulsions, lotions, gels, masks, ointments and detergents, but is not limited to the above-mentioned dosage forms. Instead, various different external preparations are produced by sterilizing the above-mentioned homogeneous polysaccharide and excipients used in the cosmetic field according to methods known in the skin care industry. be able to.

When preparing skin care cosmetics, the prepared homogeneous polysaccharide powder can be mixed with known cosmetic and pharmaceutical bases or excipients, carriers, and additives according to conventional methods. The homogeneous polysaccharide powder accounts for 3% to 10% of the total weight of the cosmetic product.

FIG. 2 is an elution curve graph of a raw polysaccharide fraction passed through a DEAE Fast Flow ion chromatography column. This is an HPGPC gel chromatogram of a homogeneous polysaccharide. This is a GC-MS total ion flow chromatogram of six types of standard monosaccharides. This is a GC-MS total ion flow chromatogram of a homogeneous polysaccharide. This is an infrared spectrum of a homogeneous polysaccharide. This is a ¹ H spectrum of a homogeneous polysaccharide. Note: (Left → Right). This is the ^13C spectrum of the homogeneous polysaccharide. Note: (Left → Right).

The present invention will be described in more detail below in conjunction with specific embodiments.

Example 1 Preparation and structural characterization of Sashigoka homogeneous polysaccharide 1. Extraction of Sashigoka crude polysaccharide:
Grind the dried Sashigoka drink powder , pass it through a 100 mesh sieve, extract the sieved medicinal powder with 5 times its weight of water at a temperature of 80℃ for 2 hours each time, 3 times in total, and extract the extract. The supernatant liquid is collected by centrifugation at 3000 rpm/min for 20 min, and the supernatant liquid is concentrated to one-fifth of the original volume by rotary evaporation to obtain a concentrated liquid. After cooling the concentrate, α-amylase was added to 0.1% by weight, the pH value was adjusted to 7.0, and the solution was exposed to iodine-potassium iodide reagent for 4 h in a 60°C water bath. Enzyme digestion is carried out (until the solution no longer changes color) and the temperature is rapidly raised to 100° C. and held for 5 min to inactivate the enzyme, followed by centrifugation at 3000 rpm/min for 10 min to collect the supernatant. The supernatant and Sevage reagent (chloroform:n-butanol mixed at a volume ratio of 4:1) were mixed at a volume ratio of 1:1, shaken vigorously for 30 minutes, and then left to stand for 12 hours to remove the polysaccharide solution in the upper layer. Collect, mix the upper polysaccharide solution and Sevage reagent in a 1:1 volume ratio, and repeat the above operation until the characteristic absorption peak of the protein disappears in the UV scan. After concentrating the finally collected upper layer polysaccharide solution, 4 times the volume of absolute ethanol was added, and the mixture was allowed to stand at 4°C for 48 hours to precipitate. Then, the precipitate was centrifuged at 3000 rpm/min for 10 min. Collect. After adding anhydrous ethanol to the precipitate and repeating the above operation three times, the finally obtained precipitate is freeze-dried to obtain a crude polysaccharide powder of Sashigoka.

2. Purification of Homogeneous Polysaccharide of Sashigoka After completely dissolving the obtained crude polysaccharide powder of Sashigoka in distilled water, it was separated using a DEAE Fast Flow ion chromatography column, and the elution conditions were as follows: a flow rate of 2.5 mL/min. min, and the solution is sequentially eluted with water, 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.3 mol/L, and 0.5 mol/L sodium chloride solutions. Collect gradients using a fully automatic collector, elute each gradient solution with 3 times the column volume, collect 30 tubes in total at 5.0 mL per tube, and perform septum tube follow-up detection using the sulfuric acid-phenol method. I do. FIG. 1 shows an elution curve graph of the Stimagoka crude polysaccharide fraction passed through a DEAE Fast Flow ion chromatography column.

On the one hand, the collected liquids corresponding to the different eluates are concentrated to a certain volume and then dialyzed using a dialysis bag (cutoff molecular weight 3500 Da). The water in the beaker containing the dialysis bag is exchanged five times a day, and after dialysis for 7 days, the polysaccharide solution in the dialysis bag is centrifuged and freeze-dried to obtain initially purified polysaccharide.

On the other hand, after concentrating the collected eluate, it was reseparated on a Sephadex G-200 dextran gel column, eluted with distilled water at a flow rate of 0.5 mL/min, and 5.0 mL/tube was collected using a fully automatic collector. After collecting 0 mL and detecting with phenol-sulfuric acid, collect the main peak part of the elution curve. Subsequently, the solution in the main peak part of the collected elution curve was concentrated, and then dialyzed for 2 days to desalt using a dialysis bag with a cut-off molecular weight of 3500 Da.Finally, the solution in the dialysis bag was concentrated and freeze-dried. , obtain a homogeneous polysaccharide powder.

3. Purity measurement of homogeneous polysaccharide HPLC conditions: Agilent 1200 high performance liquid chromatography, columns TSK GEL G3000PW _XL (7.8 x 300 mm, 7 μm) and TSK GEL G5000PW _XL (7.8 x 300 mm, 10 μm) were connected in series, the mobile phase was 0.02 mol/L KH ₂ PO ₄ solution, the flow rate was 0.5 mL/min, the column temperature was 35 °C, and the detector was a Waters 2414 differential refractive index detector. It is. The homogeneous polysaccharide obtained in Step 2 was dissolved in an appropriate amount of water to make an injection sample of 10 μL. The chromatogram shows that the chromatographic peak of the homogeneous polysaccharide is a single one, as shown in Figure 2. One symmetrical peak was observed, indicating that the homogeneous polysaccharide prepared according to the present invention is indeed a homogeneous polysaccharide.

4. Determination of the molecular weight of the homogeneous polysaccharide of staghorn polysaccharides 1 mg of pullulan polysaccharides with molecular weights of 50K, 80K, 150K, 270K, 410K and 670K Da were each dissolved in 1 mL of water and analyzed using an Agilent 1200 high-speed analyzer under the HPLC conditions described above. Analyzed by liquid chromatography, recording the retention time, with the logarithm of the relative molecular weight (logM) as the ordinate and the retention time (t) as the abscissa, yielding a standard curve y=14.62-0.980X, By substituting the peak appearance time of the homogeneous polysaccharide in the sting to the curve equation, the molecular weight of the homogeneous polysaccharide in the sting is determined as 6.83×10 ⁵ Da.

5. Structural characterization of the homogeneous polysaccharide 5.1 Analysis of the polysaccharide fraction First, acid hydrolysis is performed, followed by acetylation and derivatization, followed by gas phase GC analysis. As a method for acid hydrolysis, we weighed 10 mg of a sample of the homogeneous polysaccharide, put each into an ampoule bottle, added 4 mL of trifluoroacetic acid with a concentration of 2 mol/L, blown out the air in the bottle with nitrogen, and added alcohol. Seal the tube with a torch. After hydrolysis at 110 °C for 6 hours, the sample was dried by rotary evaporation, and 2 mL of methanol was added to dissolve and evaporate, repeated three times to remove as much trifluoroacetic acid in the sample as possible. Obtain polysaccharide hydrolyzate. After adding 1 mL of methanol and transferring the sample to a serum vial and blow drying with nitrogen, 1.0 mL of pyridine, 10 mg of hydroxylamine hydrochloride, and 1.0 mg of internal standard inositol were added to the polysaccharide hydrolyzate, and 90 A shaking reaction is carried out for 0.5 h at a constant temperature of °C, and after cooling, 1 mL of acetic anhydride is added, and an acetylation reaction is carried out at 90 °C for 0.5 h. After cooling, water is added to terminate the reaction. Further, 2.0 mL of chloroform is added and extracted three times, excess water is removed with anhydrous sodium sulfate, and the mixture is filtered through a 0.22 μm organic phase filtration membrane. Standards of each monosaccharide are also derivatized according to the steps above.

After derivatization according to the above method, GC analysis is performed. The GC detection conditions were an Agilent 6890N gas chromatography system using an Agilent HP-5 quartz capillary column (30 m x 0.32 mm x 0.25 μm), constant pressure mode at 20 PSI, carrier gas at N ₂ , and sample injection volume at 1. .0 μL, the flow rate was 1.0 mL/min, the temperature of the sample inlet was 250 °C, the temperature setting of the FID detector was 250 °C, the sample inlet was in non-split mode, and the program temperature was increased, i.e. 100 °C. After holding the initial temperature of .degree. C. for 30 seconds and raising the temperature to 160.degree. C. at a rate of 3.degree. C./min, the temperature increase is continued by changing the temperature increase rate. The temperature is raised to 250°C at a rate of 10°C/min and held for 5 min. Figure 3 shows the GC-MS total ion flow chromatogram of six types of monosaccharide standards: arabinose, galactose, glucose, mannose, xylose, and fucose. The GC-MS total ion flow chromatogram of the homogeneous polysaccharide shown in Figure 4 is shown in Figure 4.

The GC results were analyzed according to the retention time of six types of monosaccharide standards such as arabinose, galactose, glucose, mannose, xylose and fucose, and the monosaccharide mole percent of the homogeneous polysaccharide was calculated from the area ratio of peak appearance. calculate. As a result, by comparing the retention time in the GC spectrum of the monosaccharide standard, it was shown that the homogeneous polysaccharide is composed of fucose, arabinose, galactose, glucose , and xylose, and by the internal standard method, five types of The mole percentages of monosaccharides are calculated as 16.42%, 32.27%, 40.38%, 7.21% and 3.72%.

5.2 Infrared spectral scan of Sashigoka homogeneous polysaccharide 2.0 mg of Sashigoka homogeneous polysaccharide sample is weighed, mixed with potassium bromide powder, polished uniformly, and then tableted. Thereafter, the tablet was placed in a Fourier transform infrared spectrometer to perform an infrared scan (400-4000 cm ⁻¹ ), and the infrared absorption spectrum of the sample was recorded, and the results are shown in FIG. As can be seen from the IR diagram, the polysaccharide homogeneous polysaccharide exhibits typical polysaccharide absorption characteristics, where 3456 cm ^-1 and 2929 cm ^-1 are the stretching vibration peaks of O-H bonds, and 1744 cm ^-1 is the CH ₂ , 1635 cm ^-1 is the stretching vibration peak due to CO ₂ or co-produced water, 1411 cm ^-1 is the bending vibration peak of C-O bond, and 1242 cm ^-1 is the peak of primary alcohol. This is a stretching vibration peak due to β-OH. The absorption peak at 1021 cm ^-1 indicates that the homogeneous polysaccharide contains a pyran ring, and the absorption peak at 893 cm ^-1 indicates that the homogeneous polysaccharide contains β- The absorption peak at 835 cm ⁻¹ indicates that the polysaccharide contains α-glycosidic bonds.

5.3 Methylation analysis of Sashigoka homogeneous polysaccharide Weigh 20 mg of Sashigoka homogeneous polysaccharide sample (sufficiently dried sample), put it into a test tube with a stopper, and use 6 mL of dimethyl sulfoxide DMSO reagent. and after sealing with nitrogen, heating and mixing homogeneously with magnetic stirring, adding sodium hydroxide (240 mg of sodium hydroxide in 6 mL of DMSO) to form a sodium hydroxide suspension; Leave overnight. The next day, add 3.6 mL of iodomethane to the test tube, stir for 8 min, then blow out the iodomethane with nitrogen to methylate it again, repeat this 3 times, and then stop the reaction by adding 6 mL of distilled water. do. After dialysis against running water and deionized water for 24 h, it was extracted three times with chloroform, dried over anhydrous sodium sulfite for 24 h, and then blown dry with nitrogen, leaving about 1 mL of solution. After hydrolyzing with trifluoroacetic acid, 70 mg of sodium borohydride NaBH4 was added and stirred for 24 hours, and a strongly acidic anion and cation exchange resin was further added and stirred for 10 minutes to mix uniformly, followed by suction filtration. The filtrate is collected, methanol is added thereto, and the mixture is dried by blowing with nitrogen, followed by adding 0.5 mL each of acetic anhydride and anhydrous pyridine, and acetylating at 100° C. for 2 hours. After the reaction is completed, acetic anhydride is removed by repeatedly adding absolute ethanol, and further GC-MS analysis is performed.

The GC-MS chromatography conditions were an Agilent 6890-5973N gas chromatography mass spectrometer, the column was an HP-5 MS capillary column (30 m x 250 pm x 0.25 um D), the carrier gas was helium He, and the heater The temperature was 250°C, and the program temperature was raised, that is, the temperature was raised from the initial temperature to 200°C at a rate of 140°C/min, held for 5 min, and further raised to 240°C at a rate of 8°C/min, and the split ratio was The ratio is 50:1, and the sample injection volume is 5 μL. Table 1 shows the methylation analysis data of the homogeneous polysaccharide. As a result of the methylation of the Stinggoka homogeneous polysaccharide, the Stinggoka homogeneous polysaccharide is mainly composed of →,6)-β-Galp(1→, Glcp residue is the main unit of the Stinggoka homogeneous polysaccharide, It was found that they were bound in the 1→4, 1→6, 1→4,6, and 1→ forms, and the Galp residue was bound in the 1→4 form.
Table 1 Methylation analysis data of Sashigoka homogeneous polysaccharide

5.4 NMR analysis of Sashigoka homogeneous polysaccharide Weighed 30 mg of dried Sashigoka homogeneous polysaccharide, dissolved it in 0.5 ml of _D2O , heated it at 60°C for 1 hour to completely dissolve it, and then transferred it to a nuclear magnetic tube. ^{The 1} H spectrum and ¹³ C spectrum were measured using an AV300 nuclear magnetic resonance apparatus manufactured by Brucker, Germany, and the results are shown in FIGS. 6 and 7. As can be seen from the spectrum, the signal distribution range of the ¹ H spectrum of the homogeneous polysaccharide is narrow, mainly δ2.0-6.0 ppm (4.19, 4.09, 4.07, 4.00, 3.0 ppm). 95, 3.90, 3.70, 3.68, 3.65, 3.64, 3.62, 3.61, 3.60, 3.59, 3.58, 3.55, 3.54 and 3.52 ppm). On the other hand, the signal range of the C spectrum is δ60-110 ppm (107.41, 106.83, 106.36, 103.62, 95.72, 92.11, 91.85, 84.00, 81.31 , 76.81, 76.64, 76.31, 75.50, 71.24, 71.14, 71.00, 69.89, 69.21, 69.14, 62.30, 61.26, 61 .05, 60.68, and 60.42 ppm). Table 2 shows the chemical shift assignments of the anomeric carbons of individual sugar residues in the homogeneous polysaccharide.
Table 2 Chemical shift assignment of hydrocarbons in the homogeneous polysaccharide

Example 2 Evaluation of the antioxidant activity of the homogeneous polysaccharide In modern medicine, the main cause of skin aging is oxidative stress in the dermal and epidermal layers caused by various factors, so polysaccharide has a strong antioxidant effect. The substance is believed to have a better anti-skin aging effect, and in this experiment, the homogeneous polysaccharide obtained according to Example 1 was used to evaluate the antioxidant activity.

1. Measurement of DPPH radical scavenging activity Take 2 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 μg·ml ⁻¹ ), add 2 mL of DPPH solution (100 μg·ml ⁻¹ ), and stir thoroughly. After mixing, the mixture is protected from light and allowed to react for 30 minutes. The absorbance value (Am) of the reaction system is measured at 517 nm. At the same time, a solvent blank group (An, replacing the DPPH solution with an equal volume of methanol) and a sample blank group (Ao, replacing the sample solution with an equal volume of methanol) are set up. VC is used as a positive control group. The experiment is conducted three times in parallel, and the elimination rate of DPPH by the extract is calculated according to the following formula.

2. Measurement of ABTS ⁺ scavenging activity Take 0.4 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 μg ml ⁻¹ ), add 4 mL of ABTS ⁺ solution, and react for 6 min. Measure its absorbance (Ai) at 734 nm. At the same time, set up a solvent blank group (Aj, replacing the ABTS ⁺ solution with an equal volume of methanol), a sample blank group (Ah, replacing the sample solution with an equal volume of methanol) and a positive control group. The experiment is performed three times in parallel, and the elimination rate of ABTS ⁺ by the extract is calculated according to the following formula.

3. Measurement of reducing power Take 0.8 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 μg·ml ⁻¹ ), add 2 mL of phosphate buffer (PH = 6.6) and 2 mL of 1 After adding % potassium ferricyanide and water bathing at 50 °C for 20 min, 2 mL of 10% trichloroacetic acid was added, centrifuged at 3000 rpm for 10 min, 2 mL of supernatant was taken, and 2 mL of deionized water and 0.4 mL were added. After adding 0.1% ferric chloride of The larger the value, the stronger the reduction ability.

Results: As shown in Table 3-5, the IC ₅₀ values for scavenging DPPH radical and ABTS ⁺ by the homogeneous polysaccharide were 3.17 ± 0.57 μg·mL ⁻¹ and 15.26 ± 5.79 μg, respectively.・mL ^-1 , and the _EC50 value of reducing power is 36.02±10.89μg・mL ^-1 , and its antioxidant power is stronger than the positive drug vitamin C, and the difference is statistically significant. (P<0.01), indicating that the homogeneous polysaccharide prepared in Example 1 had strong antioxidant activity.

Example 3 Preparation of an essence emulsion containing a homogeneous polysaccharide obtained in Example 1 In this experiment, an essence emulsion was prepared using the homogeneous polysaccharide obtained in Example 1, and the weight percentages of the fractions and the production process were as follows. It is as follows.

Production process: Heat phase A and phase B to 70℃ each with stirring to ensure everything is dissolved and mixed uniformly, then add phase B to phase A at 70℃ to form a W/Q type emulsion. form. After uniform stirring and cooling to room temperature, the product is obtained.

The components of the base used in the preferred embodiment of the present invention are as described above, and the components of the base used in this embodiment can optimize the efficacy of the drug composition of the present invention. However, common bases manufactured by other manufacturers that are applicable to cosmetics can also be used in the present invention, and as long as the amount used meets the Chinese cosmetic additive dosage standards, it will not affect the effects of the present invention. Since it has no effect, its details are omitted here.

The homogenization/emulsification device used in the present invention is a FV-30L FISCO vacuum homogenization/emulsification machine produced by Shanghai FLUKO Fluid Machine Manufacturing Co., Ltd., and has functions such as homogenization, stirring, and temperature control. Homogenization and emulsification equipment for cosmetic preparation produced by other manufacturers can also be used in the present invention and will achieve the effects described in the present invention, provided they are operated strictly according to the process parameters described in the present invention. be able to.

Example 4 Evaluation of the anti-skin aging activity of an essence emulsion containing the homogeneous polysaccharide obtained in Example 3. In this experiment, the essence emulsion containing the homogeneous polysaccharide obtained in Example 3 was used to evaluate the anti-skin aging activity.

1 Materials and Methods 1.1 Experimental Animals 40 experimental animal SPF grade Kunming female mice with a mass of (20 ± 2) g were obtained from Guangdong Provincial Laboratory Animal Center, Laboratory Animal Quality License Number: 44005800003406, Guangzhou Traditional Chinese Medicine. The animals were kept in the animal house of the University Science and Technology Industry Park (license number: SYXK (Guangdong) 2013-0014) and experiments were conducted. Since the ultimate goal of this research is to develop anti-skin aging cosmetics suitable for women, all female mice were used as experimental animals. Mice were disposed of according to principles of animal ethics.

1.2 Materials and Reagents The essence emulsion containing the homogeneous polysaccharide was the essence emulsion prepared in Example 3, D-galactose was obtained from Beijing Solarbio Biotechnology Co., Ltd., and the HA measurement kit was obtained from Shanghai Fermention Biotechnology Co., Ltd. Science and Technology Co., Ltd., and the HYP measurement kit, SOD measurement kit, and Coomassie Brilliant Blue were obtained from Nanjing Jiancheng Biological Engineering Research Institute.

1.3 Grouping and Modeling First, female mice are adaptively housed for 7 d and experiments are performed after they are acclimatized to the current environment. Thirty female mice were divided into three groups using a random number table, two of which were used as the modeling group, and D-galactose was subcutaneously injected at 1.0 g·kg ^-1 ·d ^-1 for a total of 30 days. The remaining one group of mice served as a blank group and received daily injections of the same amount of saline. Comparing the skin appearance of the modeling group and the blank group after 30 d, skin laxity and fine wrinkles were noticeable in the model group, but the opposite was true in the blank group. Two groups of model mice with skin aging were divided into a model group and a homogeneous polysaccharide group. The hair on the mouse's back was cut short with scissors and then shaved with a razor.

1.4 Essence emulsion for external use on mouse skin The essence emulsion containing 6% of the homogeneous polysaccharide obtained in Example 3 was used for the mice in the homogeneous polysaccharide group; Using an essence emulsion that does not contain polysaccharides, specifically, a 4 cm x 7 cm area was selected at the center of the back of the mouse, and the cream of each group was applied to the surface of the selected corresponding skin area of each group of mice. 0.3 g per mouse per day; after 24 h, the skin was washed and reapplied; continued application for 21 d; manual depilation was also performed 4 times during this period.

1.5 Observation of expressive characteristics of mouse skin Expressive characteristics such as color, smoothness, and wrinkles of the skin at the drug administration site of mice in each group were compared and photographed and recorded. After that, the experimental mouse was killed by decapitation, the skin at the drug injection site was quickly peeled off, connective tissue such as subcutaneous fat was removed, and the mouse was spread out flat, and the mouse skin was cut out at the center using a punch with a diameter of 2 cm. The moisture content is measured, and the remaining skin is stored frozen at -20°C and used to measure components such as hydroxyproline in the skin.

1.6 Measurement of skin moisture content After accurately weighing the wet weight of the skin cut out with a punch, it was immediately placed in an oven and dried at 50°C for 12 hours, the dry weight was measured, and the skin moisture content of each experimental group was calculated. did. ceremony,
It is.

1.7 Measurement of the content of hydroxyproline (HYP) in the skin Take 0.5 g of skin tissue from the drug administration site, wash it with physiological saline pre-cooled in an ice bath, dry it with filter paper, and then pre-cool it in an ice bath. The mixture was added to normal saline solution and polished to a 10% homogenate using a glass homogenizer. The obtained homogenate is centrifuged at 3000 r/min for 10 min at 0°C, and the supernatant is collected. According to the method described in the instruction manual of the HYP kit, the OD value was measured using an ultramicroplate spectrophotometer, and the content of HYP in the skin of mice in each experimental group was calculated.

1.8 Measurement of hyaluronic acid (HA) content in the skin Take 0.5 g of skin tissue from the drug administration site, add it to PBS buffer pre-cooled in an ice bath, and polish to a 10% homogenate using a glass homogenizer. did. The obtained homogenate is centrifuged at 1000 r/min for 4 minutes at 0°C, and the supernatant is collected. According to the method described in the instruction manual of the HA kit, the OD value was measured using an ultra-microplate spectrophotometer, and the content of HA in the skin of mice in each experimental group was calculated.

1.9 Measurement of superoxide dismutase (SOD) activity in the skin Take 0.5 g of skin tissue from the drug administration site, wash it with physiological saline pre-cooled in an ice bath, and add it to the physiological saline pre-cooled in an ice bath. , and polished to a 10% homogenate using a glass homogenizer. The obtained homogenate is centrifuged at 3000 r/min for 10 min at 0°C, and the supernatant is collected. According to the method described in the instruction manual of the SOD kit, the OD value was measured using an ultra-trace microplate spectrophotometer, and the activity of SOD in the skin of mice in each experimental group was calculated.

1.10 Regarding statistical methods, SPSS 18.0 statistical software was used to analyze the data, and data calculations were performed using SPSS18.0 statistical software.
One-way analysis of variance was used for between-group comparisons, and Dunnett T3 test was used if the results of the homogeneity of variance test were significant, and LSD test was used if the results of the homogeneity of variance test were not significant. Pairwise comparisons were made using , and a statistically significant difference was determined at P<0.05.

2 Results 2.1 Comparison of skin appearance When the skin of mice in each group was observed, it was found that mice in the model group had looser skin, more wrinkles, and slower body hair regeneration than animals in the blank group. . Mice coated with 6% homogeneous polysaccharide showed clear improvement in skin wrinkles, smoothness, and laxity compared to the model group.

2.2 Effects of staghorn homogeneous polysaccharide on skin moisture content, HYP content, HA content, and SOD activity in mice Measurement of water content, HYP content, HA content, and SOD activity in skin tissue of three groups of mice The results are shown in Table 6. Moisture content, HYP content, HA content, and SOD activity in the skin tissue of mice in the model group were clearly lower than those in the blank group (P<0.01), indicating that the mouse skin aging model was successfully constructed. It has been shown. Compared with the model group, the moisture content, HYP content, HA content, and SOD activity of the skin of mice in the homogeneous polysaccharide group were clearly increased, and statistically significant differences were observed (P <0.01). Compared with the blank group, there were no significant differences in the skin moisture content, HYP content, HA content, SOD activity, and other indicators of the mice in the homogeneous polysaccharide group (P>0.01).

Compared with the model group, ^△ P<0.05, ^△△ P<0.01.

In summary, as a result of this experiment, we found that the mice in the blank group had smooth, firm, and elastic skin. In skin aging model mice injected with D-galactose, the skin becomes loose and wrinkles increase, and indicators such as skin moisture content, hydroxyproline content, hyaluronic acid content, and superoxide dismutase activity significantly increase. It declined to . As a result of measurement, when model mice were externally applied with an essence emulsion containing 6% homogeneous polysaccharide, the skin appearance was clearly improved, and the moisture content, hyaluronic acid content, and hydroxyproline content of the mouse skin were significantly improved. Indices such as the content of superoxide dismutase and superoxide dismutase activity were also clearly improved.

Finally, in this specification, the above examples are only used to further explain the technical solutions of the present invention, and should not be understood as limiting the protection scope of the present invention. It is necessary to mention that some non-essential improvements and adjustments made by those skilled in the art according to the above description of the present invention fall within the protection scope of the present invention.

Claims (6)

1) After crushing the dried Sashigoka drink pieces and passing them through a sieve, add 5 to 8 times the amount of water to the medicinal powder, and extract at a temperature of 80 to 100°C for 2 hours each time, 3 times in total to prepare the extract. combining, centrifuging to collect the supernatant, and concentrating the supernatant to obtain a concentrate;
2) After cooling the concentrate, add α-amylase to a weight of 0.1 to 0.4%, adjust the pH value to 7.0, and add iodine-iodine in a water bath at 60°C. Exposing the solution to a potassium reagent to perform enzymatic decomposition until the solution no longer changes color, rapidly raising the temperature to 100°C and holding for 5 minutes to inactivate the enzyme, and collecting the supernatant by centrifugation;
3) Mix the supernatant liquid collected in step 2) and Sevage reagent at a volume ratio of 1:1, shake vigorously for 30 min, let stand for 12 h, collect the upper layer polysaccharide solution, and mix with the upper layer polysaccharide solution and Sevage reagent. The reagents were mixed in a volume ratio of 1:1 and the above operation was repeated until the characteristic absorption peak of the protein disappeared in the UV scan.The Sevage reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The steps you can take and
4) After concentrating the upper polysaccharide solution finally collected in step 3), add 4 to 6 times the volume of absolute ethanol, let it stand at 4°C for 48 hours to precipitate it, and then centrifuge it. collecting the precipitate, adding absolute ethanol to the precipitate, repeating the above operation three times, and then freeze-drying it to obtain a crude polysaccharide powder of Sashigoka;
5) After completely dissolving the crude polysaccharide powder of Sashigoka obtained in step 4) in distilled water, it was separated using a DEAE Fast Flow ion chromatography column, and the elution conditions were a flow rate of 2.5 mL/min. , pure water, 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 1 mol/L sodium chloride solution, followed by follow-up detection using the sulfuric acid-phenol method. performing and collecting the eluate;
6) After concentrating the eluate collected in step 5), it was reseparated using a Sephadex G-200 dextran gel column, eluted with distilled water at a flow rate of 0.5 mL/min, and detected using the phenol-sulfuric acid method. collecting a main peak portion of the elution curve;
7) After concentrating the solution in the main peak part of the elution curve collected in step 6), it is desalted by dialysis for 2 days using a dialysis bag with a cut-off molecular weight of 3500 Da, and finally the solution in the dialysis bag is concentrated. A method for preparing a sashigoka polysaccharide, comprising the step of freeze-drying to obtain a polysaccharide powder . 1) Grind the dried sashigoka drink and pass it through a 100 mesh sieve.The sieved medicinal powder is mixed with 5 to 8 times its weight of water at a temperature of 80 to 100°C for 2 hours each time, 3 times in total. extracting, combining the extracts, centrifuging at 3000 rpm/min for 20 min to collect the supernatant, and concentrating the supernatant to one-fifth of the original volume by rotary evaporation to obtain a concentrate;
2) After cooling the concentrate, add α-amylase to a weight of 0.1 to 0.4%, adjust the pH value to 7.0, and perform enzymatic decomposition in a 60°C water bath for 4 hours. At this time, the solution no longer changes color when exposed to the iodine-potassium iodide reagent, and then the temperature is rapidly raised to 100°C and held for 5 minutes to inactivate the enzyme, followed by centrifugation at 3000 rpm/min for 10 minutes. and collecting the supernatant;
3) Mix the supernatant liquid collected in step 2) and Sevage reagent at a volume ratio of 1:1, shake vigorously for 30 min, let stand for 12 h, collect the upper layer polysaccharide solution, and mix with the upper layer polysaccharide solution and Sevage reagent. Mix the reagents in a 1:1 volume ratio and repeat the above operation until the characteristic absorption peak of the protein disappears in the UV scan, and the Sevage reagent mixes chloroform and n-butanol in a 4:1 volume ratio a step manufactured by
4) After concentrating the upper layer polysaccharide solution finally collected in step 3), add 4 to 6 times the volume of absolute ethanol, let it stand at 4°C for 48 hours to precipitate it, and then turn the solution at 3000 rpm/min. Collect the precipitate by centrifugation for 10 min, add absolute ethanol to the precipitate, repeat the above operation three times, freeze-dry the final precipitate, and prepare the crude polysaccharide of sashigoka. obtaining a powder;
5) After completely dissolving the crude polysaccharide powder of Sashigoka obtained in step 4) in distilled water, it was separated using a DEAE Fast Flow ion chromatography column, and the elution conditions were a flow rate of 2.5 mL/min. , pure water, 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 1 mol/L sodium chloride solution, and then the eluate was collected using a fully automatic collector. Collect 30 tubes with a gradient, elute each gradient solution with 3 column volumes, 5.0 mL per tube, and perform septum tube follow-up detection using the sulfuric acid-phenol method. and,
6) After concentrating the eluate collected in step 5), it was reseparated using a Sephadex G-200 dextran gel column, eluted with distilled water at a flow rate of 0.5 mL/min, and collected per tube using a fully automatic collector. collecting the main peak portion of the elution curve after detecting with phenol-sulfuric acid;
7) After concentrating the solution in the main peak part of the elution curve collected in step 6), it is desalted by dialysis for 2 days using a dialysis bag with a cut-off molecular weight of 3500 Da, and finally the solution in the dialysis bag is concentrated. A method for preparing a stinging polysaccharide having anti-skin aging effect, characterized in that it comprises the step of freeze-drying to obtain a polysaccharide powder . A stricken polysaccharide having an anti-skin aging effect obtained by the method for preparing a stricken polysaccharide having an anti-skin aging effect according to claim 1 or 2 . Use of the stingogen polysaccharide according to claim 3 in the manufacture of skin care cosmetics or therapeutic agents for the skin having anti-skin aging effects. A skin care cosmetic product, comprising the polysaccharide according to claim 3 and an excipient used in the field of cosmetics. A dermatological therapeutic agent comprising a stingogen polysaccharide according to claim 3 and a medically acceptable carrier.