Preparation method and application of pittosporum illicioides polysaccharide

Abstract

The application discloses a preparation method of a PITTOSPORUM glabratum polysaccharide, and the method comprises the following steps: taking washed and dried PITTOSPORUM glabratum leaves, crushing the leaves, and then screening the crushed leaves through a 40-mesh sieve; adding 95% ethanol into the screened leaves according to a material-liquid ratio of 1 / 20 (g / mL) to remove impurities through hot reflux for 6 hours; and airing the filtered residue; adding deionized water into the filtered residue of step S1 according to a material-liquid ratio of 1 / 25 (g / mL), and heating and boiling the residue to extract the residue for 6 hours; filtering and collecting the extracted liquid, and reducing and concentrating the extracted liquid; collecting the precipitate of the solution after the step S2 by suction filtration, drying the solvent, and then redissolving the precipitate in deionized water; removing protein from the redissolved solution by using a Sevage method; and freeze-drying the solution to obtain the PITTOSPORUM glabratum polysaccharide.

Description

Technical Field

[0001] This invention relates to the technical field of cosmetic additives with antioxidant, whitening, and anti-wrinkle properties, specifically to a method for preparing Pittosporum tobira polysaccharide and its application. Background Technology

[0002] Pittosporum tobira ( Pittosporum glabratum *Pittosporum* (Lindl.) is a plant belonging to the genus *Pittosporum* in the family Pittosporaceae. It is also known as Long-fruited Fragrant Pittosporum, One-Cloud Pittosporum, and Glossy Pittosporum. The roots and leaves of *Pittosporum glabra* have a long history of use in traditional Chinese medicine, possessing properties that tonify the lungs and kidneys, dispel wind and invigorate the meridians, reduce swelling and detoxify, and promote blood circulation and relieve pain. It is commonly used to treat conditions such as consumptive cough and asthma, sciatica, rheumatoid arthritis, and sequelae of poliomyelitis. In recent years, research on the saponins and triterpenoids in *Pittosporum glabra*, both domestically and internationally, has deepened, revealing its high medicinal value in antiviral, analgesic, and hemostatic effects.

[0003] The use of traditional Chinese herbal medicines for beauty, both internally and externally, has a history of thousands of years in China, boasting advantages such as convenient sourcing, high safety, low irritation, and stable medicinal properties. In contemporary society, various cosmetics are emerging in endless streams, but most of these beauty and cosmetic products are made of chemical substances and have certain toxic side effects. Therefore, with the increasing trend of valuing natural products, non-toxic, pollution-free, and side-effect-free natural skincare ingredients are becoming increasingly popular among consumers. Pittosporum tobira is widely cultivated in 18 provinces of my country, with abundant resources and low prices. If its leaf polysaccharides can be successfully applied to the field of cosmetics with whitening and anti-wrinkle effects, they can achieve deeper development and utilization.

[0004] There is very little research on polysaccharides in Pittosporum tobira, and no reports have been found on their antioxidant, whitening, and anti-wrinkle activities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for preparing Pittosporum glabra polysaccharide and its application. The Pittosporum glabra polysaccharide obtained by this invention is extracted from Pittosporum glabra leaves and has significant inhibitory effects on DPPH and OH free radicals, tyrosinase, and elastase.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing polysaccharides from Pittosporum tobira, the method comprising the following steps:

[0008] S1 Take cleaned and dried Pittosporum tobira leaves, crush them and pass them through a 40-mesh sieve. Add 95% ethanol at a material-to-liquid ratio of 1 / 20 (g / mL) and reflux for 6 hours to remove impurities. Let the filter residue dry.

[0009] S2 Add the dried Pittosporum tortoiseshell leaves from step S1 to deionized water at a material-to-liquid ratio of 1 / 25 (g / mL), heat to boiling and extract for 6 h, filter to collect the extract and concentrate under reduced pressure.

[0010] S3. The solution concentrated under reduced pressure in step S2 is filtered to collect the precipitate. After evaporating the solvent, it is redissolved in deionized water, deproteinized using the Sevage method, and freeze-dried to obtain the Pittosporum glabra polysaccharide.

[0011] It should be noted that in step S2, during concentration, the concentrate is precipitated with 4 times the volume of 95% ethanol at 4°C for 12 hours.

[0012] It should be noted that in the filtration and collection of precipitate in step S3, the precipitate is washed three times in sequence with 95% ethanol, anhydrous ethanol, acetone, and diethyl ether, with 1g of precipitate corresponding to 50 mL of solvent.

[0013] One solution provided by this invention:

[0014] A polysaccharide of Pittosporum glabra obtained by a preparation method of Pittosporum glabra polysaccharide, which is then applied to cosmetics for its antioxidant, whitening and anti-wrinkle effects.

[0015] The present invention proposes another solution:

[0016] A cosmetic product comprising Pittosporum tobira polysaccharide obtained by a preparation method thereof, wherein the Pittosporum tobira polysaccharide is one of the cosmetic additives.

[0017] The beneficial effect of this invention is that a polysaccharide was prepared from the leaves of Pittosporum glabra. Test results show that the Pittosporum glabra polysaccharide has high antioxidant activity and high inhibitory effect on tyrosinase and elastase. Therefore, it can be used to develop into a natural cosmetic additive with antioxidant, whitening and anti-wrinkle effects. Detailed Implementation

[0018] The present invention will be further described below. It should be noted that the following embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to these embodiments.

[0019] This invention relates to a method for preparing polysaccharides from Pittosporum tobira, the method comprising the following steps:

[0020] S1 Take cleaned and dried Pittosporum tobira leaves, crush them and pass them through a 40-mesh sieve. Add 95% ethanol at a material-to-liquid ratio of 1 / 20 (g / mL) and reflux for 6 hours to remove impurities. Let the filter residue dry.

[0021] S2 Add the dried Pittosporum tortoiseshell leaves from step S1 to deionized water at a material-to-liquid ratio of 1 / 25 (g / mL), heat to boiling and extract for 6 h, filter to collect the extract and concentrate under reduced pressure.

[0022] S3. The solution concentrated under reduced pressure in step S2 is filtered to collect the precipitate. After evaporating the solvent, it is redissolved in deionized water, deproteinized using the Sevage method, and freeze-dried to obtain the Pittosporum glabra polysaccharide.

[0023] It should be noted that in step S2, during concentration, the concentrate is precipitated with 4 times the volume of 95% ethanol at 4°C for 12 hours.

[0024] It should be noted that in the filtration and collection of precipitate in step S3, the precipitate is washed three times in sequence with 95% ethanol, anhydrous ethanol, acetone, and diethyl ether, with 1g of precipitate corresponding to 50 mL of solvent.

[0025] One solution provided by this invention:

[0026] A polysaccharide of Pittosporum glabra obtained by a preparation method of Pittosporum glabra polysaccharide, which is then applied to cosmetics for its antioxidant, whitening and anti-wrinkle effects.

[0027] The present invention proposes another solution:

[0028] A cosmetic product comprising Pittosporum tobira polysaccharide obtained by a preparation method thereof, characterized in that the Pittosporum tobira polysaccharide is one of the cosmetic additives therein.

[0029] Example 1

[0030] I. Antioxidant effects of Pittosporum glabra polysaccharides

[0031] 1. Scavenging activity of Pittosporum glabra polysaccharide against DPPH

[0032] (1) Preparation of DPPH test solution: Accurately weigh 8.0 mg of DPPH, dissolve it in 100 mL of methanol to prepare a 0.08 mg / mL DPPH solution, store it in the dark for later use.

[0033] (2) Experimental procedure: The polysaccharide prepared in Example 1 and the positive control drug butylated hydroxytoluene (BHT) were prepared into sample solutions with different concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL. 2.0 mL of each polysaccharide solution and 1.0 mL of DPPH solution were mixed and recorded as solution s; 2.0 mL of each polysaccharide solution and 1.0 mL of methanol were mixed and recorded as solution b; 2.0 mL of methanol and 1.0 mL of DPPH solution were mixed and recorded as solution c. All solutions were placed at room temperature in the dark on a shaker for 30 min, and the absorbance value A was measured at 517 nm. s A b A c Each group had 3 replicates, and the experiment was repeated 3 times. The clearance rate (%) was calculated using the formula: 100 × [1 - (A)] s -A b ) / A c The DPPH free radical scavenging rate was calculated, and the inhibition rate was plotted against the inhibitor concentration. The IC50 values ​​of Pittosporum tobira polysaccharide and the positive antioxidant BHT were determined based on the inhibition curve. 50 Values. The results are shown in Table 1.

[0034] II. Scavenging activity of Pittosporum glabra polysaccharide against OH

[0035] The polysaccharide from Pittosporum tobira prepared in Example 1 and the positive control drug VC were prepared into a sample solution of 200 μg / mL. Five stoppered test tubes were used, and 1.0 mL of 2.0 mmol / L FeSO4 and 1.0 mL of 6.0 mmol / L salicylic acid-ethanol solution were added to each tube respectively. The tubes were shaken and mixed. Then, 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the polysaccharide solution were added respectively, followed by 1, 0.8, 0.6, 0.4, 0.2, and 0.0 mL of distilled water, and then 1.0 mL of 1.0 mmol / L H2O2. The tubes were shaken and mixed, and heated in a 37 ℃ water bath for 30 min. The absorbance was measured at 510 nm using a UV spectrophotometer with distilled water as the zero point. The OH scavenging rate was calculated using the following formula: Scavenging rate (%) = 100 × [1 - (A s -A b ) / A c ], where A c A is the absorbance value after adding 1 mL of distilled water. s To add absorbance values ​​for samples of different concentrations, A b The absorbance value is without H2O2. Simultaneously, an inhibition rate was plotted against the inhibitor concentration, and the IC50 values ​​of Pittosporum tobira leaf polysaccharide and the positive antioxidant vitamin C were determined based on the inhibition curve. 50 Values. The results are shown in Table 1.

[0036] The results show that Pittosporum glabra polysaccharide has strong antioxidant activity and can be further developed into a natural antioxidant.

[0037] Table 1 Antioxidant activity of Pittosporum glabra polysaccharides

[0038]

[0039] Example 2

[0040] Whitening activity of Pittosporum tobira polysaccharide

[0041] Inhibitory effect of Pittosporum glabra polysaccharide on tyrosinase

[0042] The polysaccharide from Pittosporum tobira prepared in Example 1 and the positive control drug kojic acid were prepared into sample solutions with different concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL, respectively. The reaction was carried out in a 96-well plate with a total reaction volume of 200 μL: 40 μL of sample solution; 80 μL of 67 mmol / L phosphate buffer; 40 μL of 100 U / mL tyrosinase solution; and 40 μL of 2.5 mmol / L L-DOPA. Only 200 μL of 67 mmol / L phosphate buffer was added to the zeroing well. After the samples were added, they were mixed and incubated in a 37 ℃ water bath for 20 min. The specific absorbance of the reaction product at a wavelength of 490 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader, and the test was repeated three times.

[0043] The inhibitory effect of the sample and kojic acid on tyrosinase was calculated using the following formula: Inhibition rate (%) = 100 × [1 - (A1 - A2) / (A3 - A4)], where A1, A2, A3, and A4 are the absorbance of the sample group, sample background group (PBS instead of tyrosinase), blank group (distilled water instead of sample), and blank background group (distilled water instead of sample, PBS instead of tyrosinase), respectively. Simultaneously, the inhibition rate was plotted against the inhibitor concentration, and the IC50 values ​​of Pittosporum tobira polysaccharide and kojic acid were determined based on the inhibition curve. 50 The values ​​are shown in Table 2.

[0044] Example 3

[0045] Anti-wrinkle activity of Pittosporum tobira polysaccharide

[0046] Inhibitory effect of Pittosporum glabra polysaccharide on elastase

[0047] The polysaccharide from Pittosporum tobira prepared in Example 1 and the positive control drug oleanolic acid were prepared into sample solutions with different concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL and 25 μg / mL, respectively. The reaction was carried out in a 96-well plate. The total reaction system was 200 μL: 50 μL sample solution; 100 μL 10 mmol / L Tris buffer (pH=8.0); 25 μL 500 U / mL elastase solution; 2 mmol / L n-succinyl- α - α - α - Add 25 μL of p-nitroaniline solution. Add only 200 μL of 10 mmol / L Tris buffer to the zeroing well. After adding the sample, mix well and incubate in a 25 ℃ water bath for 20 min. Detect the specific absorbance of the reaction product at a wavelength of 410 nm using an ELISA reader. Repeat the test 3 times.

[0048] The inhibitory effects of the sample and oleanolic acid on elastase were calculated using the following formula: Inhibition rate (%) = 100 × [1 - (A1 - A2) / (A3 - A4)], where A1, A2, A3, and A4 are the absorbance of the sample group, sample background group (Tris replacing elastase), blank group (distilled water replacing sample), and blank background group (distilled water replacing sample, Tris replacing elastase), respectively. Simultaneously, the inhibition rate was plotted against the inhibitor concentration, and the IC50 values ​​of Pittosporum tobira polysaccharide and oleanolic acid were determined based on the inhibition curve. 50 The values ​​are shown in Table 2.

[0049] Table 2. Whitening and anti-wrinkle activities of Pittosporum tobira polysaccharides

[0050]

[0051] For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the protection scope of the claims of this invention.

Claims

1. A method for preparing polysaccharides from Pittosporum tobira, characterized in that, The method includes the following steps: S1 Take cleaned and dried Pittosporum tobira leaves, crush them and pass them through a 40-mesh sieve. Add 95% ethanol at a material-to-liquid ratio of 1 / 20 g / mL and reflux for 6 hours to remove impurities. Let the filter residue dry. S2 Add the dried Pittosporum tortoiseshell leaves from step S1 to deionized water at a material-to-liquid ratio of 1 / 25 g / mL, heat to boiling and extract for 6 h, filter to collect the extract and concentrate under reduced pressure. S3. The solution concentrated under reduced pressure in step S2 is filtered to collect the precipitate. After evaporating the solvent, it is redissolved in deionized water, deproteinized using the Sevage method, and freeze-dried to obtain the Pittosporum glabra polysaccharide.

2. The method for preparing Pittosporum tobira polysaccharide according to claim 1, characterized in that, In step S2, during concentration, the concentrate is precipitated with 4 times its volume of 95% ethanol at 4°C for 12 h.

3. The method for preparing Pittosporum tobira polysaccharide according to claim 1, characterized in that, In step S3, during the filtration and collection of the precipitate, the precipitate is washed three times in sequence with 95% ethanol, anhydrous ethanol, acetone, and diethyl ether, with 1g of precipitate corresponding to 50 mL of solvent.

4. A polysaccharide of Pittosporum glabra obtained by the preparation method of Pittosporum glabra according to claim 1, for use in cosmetics for anti-oxidation, whitening and anti-wrinkle purposes.

5. A cosmetic product comprising Pittosporum glabra polysaccharide obtained by the preparation method of Pittosporum glabra polysaccharide according to claim 1, characterized in that, The polysaccharide from Pittosporum tobira is one of the cosmetic additives mentioned.

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