Camellia oleifera branch polysaccharide and its application

Abstract

The present invention discloses a polysaccharide from oil-tea camellia branches, which is prepared by the following method: adding anhydrous ethanol to dried and crushed oil-tea camellia branches, heating and refluxing, then mixing with deionized water for extraction, then collecting the supernatant by solid-liquid separation, performing protein removal and dialysis treatment, and drying to obtain the oil-tea camellia branch polysaccharide. The extraction rate obtained under the extraction conditions of the present invention rises to about 2%, which greatly improves the yield. Moreover, compared with the three-phase extraction method commonly used on oil-tea camellia flowers and leaves, a lot of chemical waste materials will be generated. Moreover, the extraction method adopted will not cause excessive waste of chemical reagents. Moreover, the use of oil-tea camellia branches for polysaccharide extraction is different from the common waste of direct incineration or burial, thereby greatly improving the recovery value and utilization value of oil-tea camellia branches.

Description

Technical Field

[0001] The invention belongs to the technical field of comprehensive utilization of camellia oleifera resources, and particularly relates to a camellia oleifera branch polysaccharide and application thereof. Background Art

[0002] Camellia oleifera is a small tree or evergreen shrub in the family Theaceae, primarily found in Asia. Due to its high oil yield, the oil has become a key edible oil resource in China, spurring an increase in the cultivated area to 4.5 million hectares. Each year, Camellia oleifera trees require extensive branch pruning to increase fruit yields and, therefore, oil production.

[0003] However, current methods for disposing of tea tree branches primarily involve burning or burying them, which results in air pollution and wood waste. Different types of camellia polysaccharides are commonly extracted from camellia flowers, leaves, husks, and seed cakes. These polysaccharides vary in yield, structure, and pharmacological activity against various diseases. For example, polysaccharides from camellia husks exert antioxidant activity by increasing superoxide dismutase expression mediated by DAF-16 activation. Polysaccharides extracted from camellia flowers and leaves exhibit certain antibacterial and antioxidant activities. Polysaccharides from camellia leaves exhibit antioxidant activity, while polysaccharides from camellia seed cakes possess antioxidant and antitumor activities. However, camellia branches, a byproduct of camellia fruit production, are not effectively utilized and have resulted in significant accumulation. These wood products are typically disposed of through burying or direct incineration, which pollutes the environment and negates the medicinal value of the branches. In the present invention, polysaccharides are extracted from tea oil branches, which not only overcomes the common low extraction rate of wood in the field of polysaccharides. Under common experiments, the extraction rate of tea oil wood polysaccharides is less than 0.2%. In addition, the biological activity of crude polysaccharides from tea oil branches is explored, and it is found that they have not only common antioxidant activity, but also good anti-inflammatory activity. Summary of the Invention

[0004] The object of the present invention is to overcome at least one deficiency of the prior art and provide a method for extracting polysaccharides from oil-tea camellia branches.

[0005] The technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a Camellia oleifera branch polysaccharide prepared by the following method:

[0007] 1) Adding anhydrous ethanol to the dried and crushed camellia oleifera branches, heating under reflux, and drying to obtain camellia oleifera branch powder;

[0008] 2) Mixing the camellia oleifera branch powder obtained in step 1) with deionized water and performing extraction at an extraction temperature of 60-90°C for 60-180 minutes, followed by solid-liquid separation, collecting the supernatant, and concentrating the supernatant, adding anhydrous ethanol, precipitating, and drying to obtain a camellia oleifera branch water extract;

[0009] 3) adding water to dissolve the aqueous extract of the Camellia oleifera branches obtained in step 2) to obtain an aqueous extract solution, then adding a deproteinizing agent to collect the supernatant, and repeating the deproteinizing operation 4 to 6 times to obtain a deproteinized solution;

[0010] 4) The deproteinized solution obtained in step 3) is dialyzed to a molecular weight cut-off of 3000-4000 Da, and dried to obtain the Camellia oleifera branch polysaccharide.

[0011] In some examples, the ratio of Camellia oleifera branches to anhydrous ethanol in step 1) is 1: (9-12) g / mL.

[0012] In some examples, the material-liquid ratio of the camellia oleifera branch powder to deionized water in step 2) is 1:(10-40) g / mL.

[0013] In some examples, the material-liquid ratio of the camellia oleifera branch powder to deionized water in step 2) is 1:40 g / mL.

[0014] In some examples, the extraction time in step 2) is 120 minutes.

[0015] In some examples, the extraction temperature in step 2) is 90°C.

[0016] In some examples, the reflux temperature in step 1) is 80-100°C.

[0017] In some examples, the deproteinization reagent in step 3) is chloroform and n-butanol in a ratio of (3.9-4.1):1.

[0018] In some examples, the volume ratio of the protein removal reagent to the water extract solution in step 3) is (3.9-4.1):1.

[0019] In a second aspect, the invention provides a method for preparing an anti-inflammatory drug using the polysaccharide from Camellia oleifera branches provided in the first aspect.

[0020] The beneficial effects of the present invention are:

[0021] The extraction rate obtained under the extraction conditions of the present invention rises to about 2%, greatly improving the yield. Furthermore, compared to the three-phase extraction method commonly used on camellia flowers and leaves, which produces a lot of chemical waste materials, the adopted extraction method does not cause excessive waste of chemical reagents. Furthermore, using camellia branches for polysaccharide extraction is different from the common direct incineration or landfill waste, thereby greatly improving the recovery value and utilization value of camellia branches.

[0022] Furthermore, by testing the effect of 1 mg / mL polysaccharide content on the expression of inflammatory factors in RAW.264.7 cells under LPS interference, it was found that crude polysaccharide from oil-tea camellia branches had a significant downregulation effect on three pro-inflammatory factors, indicating that crude polysaccharide from oil-tea camellia branches has a certain anti-inflammatory regulatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The response surface analysis results of the embodiment are shown in FIG.

[0024] Figure 2 To study the anti-inflammatory activity of polysaccharides from Camellia oleifera branches. DETAILED DESCRIPTION

[0025] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention. Example

[0026] A method for extracting polysaccharides from oil-tea camellia branches in this embodiment comprises the following steps:

[0027] 1) Dry the oil-tea camellia branches in a constant temperature drying oven at 55°C for 24 hours and crush them;

[0028] 2) adding anhydrous ethanol to the camellia oleifera branches at a ratio of 1:(9-12) g / mL, refluxing at 80-100 degrees Celsius to remove fat-soluble impurities, and drying the powder at a constant temperature of 55 degrees Celsius to obtain camellia oleifera branch powder;

[0029] 3) The powder obtained in step (2) was mixed with deionized water at a material-liquid ratio of 1:(10-40) g / mL, stirred and boiled at (60-90) °C for (60-180) minutes, cooled, and centrifuged to separate the solid and liquid and collect the supernatant;

[0030] 4) Concentrating the supernatant in step (3) under reduced pressure to a quarter of its original volume to obtain a concentrated solution;

[0031] 5) adding anhydrous ethanol to the concentrated solution in step (4) to produce a flocculent precipitate, separating the solid and liquid and drying to obtain a water extract product of the oil-tea camellia branches; then adding deionized water to fully dissolve the product to obtain a water extract solution;

[0032] 6) Prepare a deproteinizing agent with chloroform and n-butanol in a ratio of 4:1. Mix the deproteinizing agent at a volume 4 times that of the aqueous extract solution in step (5) and collect the supernatant solution in a centrifuge tube. Repeat the deproteinizing operation 4-6 times until the protein precipitate is removed to obtain a deproteinized solution.

[0033] 7) The deproteinized solution in step (6) was dialyzed with pure water in a 3500Da dialysis bag for 48 hours to obtain a polysaccharide dialysate;

[0034] 8) Dry the polysaccharide dialyzate in step (7) for 72 hours to obtain Camellia oleifera branch polysaccharide powder.

[0035] The boiling point of the anhydrous ethanol used in step (2) is 78.5°C, and the reflux time is continuously 24 hours at 80-100°C until it becomes transparent and colorless.

[0036] In step (6), the centrifugal speed for separating the precipitate is 4000 rpm, the centrifugal time is greater than 10 minutes, and the other centrifugal separation and precipitation parameters are also kept consistent.

[0037] Wherein, the drying method adopted in step (8) is vacuum freeze drying.

[0038] The generated 3D response surface and 2D contour plots are shown in Figure 1 As shown in Figure 2, the relationship and interaction between these process variables are revealed. Figure 1 , B and C) resulted in an initial increase in CCBP production ( Figure 1 , EF), and then gradually decreased. Under the extraction conditions of heating temperature 90℃ and heating time 120 minutes, the yield of crude polysaccharides from oil tea branches reached the maximum. Figure 1 The results showed that the interaction between extraction temperature and extraction time was stronger than other interaction variables. Therefore, under the extraction conditions of this experimental design, we can obtain the maximum yield of polysaccharides from Camellia oleifera branches.

[0039] Table 1 Variance analysis of the response surface quadratic model for crude polysaccharide yield in Camellia oleifera branches

[0040]

[0041] The optimal extraction conditions for this hot-water extraction system for polysaccharides from Camellia oleifera branches were as follows: a solid-to-liquid ratio of 1:40.187 (g / ml), an extraction temperature of 90.77°C, and an extraction time of 131.553 minutes. Under these optimal conditions, the predicted yield of crude polysaccharides from Camellia oleifera branches was 1.935%. Considering realistic experimental conditions, this experiment used conditions close to the predicted standard (solid-to-liquid ratio of 1:40, 90°C, and 130 minutes), resulting in a final extraction yield of 1.901±0.02% of the polysaccharides from Camellia oleifera branches. This yield was close to the predicted value. Furthermore, compared to other commonly used Camellia oleifera polysaccharide extraction systems, which achieve an extraction yield of approximately 0.5% at a solid-to-liquid ratio of 1:30, a temperature of 80°C, and an extraction time of 100 minutes, our system significantly improved this yield.

[0042] The phenol-sulfuric acid method was then used to calculate total sugar content, the Bradford method to calculate protein content, and the carbazole sulfate method to calculate uronic acid content. Standard samples used included glucose, bovine serum albumin (BSA), and D-galacturonic acid. The table shows a total sugar content of 27.51%, while the protein content was a low 4.15%. The uronic acid content was 25.3%, suggesting that the Camellia oleifera branch polysaccharide may be an acidic polysaccharide.

[0043] Table 2 Contents of crude polysaccharides in Camellia oleifera branches

[0044]

[0045] The expression of inflammatory factors in RAW.264.7 cells under LPS interference was tested by testing the effect of 1 mg / mL polysaccharide content, in which the positive drug group was dexamethasone and the dosage of polysaccharide was 1 mg / mL. Figure 2 It can be analyzed that the crude polysaccharide from Camellia oleifera branches has a significant down-regulating effect on three pro-inflammatory factors, indicating that the crude polysaccharide from Camellia oleifera branches has a certain anti-inflammatory regulatory effect.

[0046] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A polysaccharide from oil-tea camellia branches, characterized in that Prepared by the following method: 1) Adding anhydrous ethanol to the dried and crushed camellia oleifera branches, heating under reflux, and drying to obtain camellia oleifera branch powder; 2) Mixing the camellia oleifera branch powder obtained in step 1) with deionized water and performing extraction at an extraction temperature of 60-90°C for 60-180 minutes, followed by solid-liquid separation, collecting the supernatant, and concentrating the supernatant, adding anhydrous ethanol, precipitating, and drying to obtain a camellia oleifera branch water extract; 3) adding water to dissolve the aqueous extract of the Camellia oleifera branches obtained in step 2) to obtain an aqueous extract solution, then adding a deproteinizing agent to collect the supernatant, and repeating the deproteinizing operation 4 to 6 times to obtain a deproteinized solution; 4) The deproteinized solution obtained in step 3) is dialyzed to a molecular weight cut-off of 3000-4000 Da, and dried to obtain the Camellia oleifera branch polysaccharide.

2. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that In the step 1), the ratio of the camellia oleifera branches to anhydrous ethanol is 1: (9-12) g / mL.

3. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that In the step 2), the material-liquid ratio of the camellia oleifera branch powder to deionized water is 1: (10-40) g / mL.

4. The Camellia oleifera branch polysaccharide according to claim 3, characterized in that In the step 2), the material-liquid ratio of the camellia oleifera branch powder to deionized water is 1:40 g / mL.

5. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that The extraction time in step 2) is 120 minutes.

6. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that The extraction temperature in step 2) is 90°C.

7. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that The reflux temperature in step 1) is 80-100°C.

8. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that The deproteinizing reagent in step 3) is chloroform and n-butanol in a ratio of (3.9-4.1):

1.

9. The Camellia oleifera branch polysaccharide according to claim 1, characterized in that In step 3), the volume ratio of the protein removal reagent to the water extract solution is (3.9-4.1):

1.

10. Use of the Camellia oleifera branch polysaccharide according to any one of claims 1 to 9 in the preparation of anti-inflammatory regulating drugs.

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