Camellia oleifera shell polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural products, and discloses camellia oleifera shell polysaccharide as well as a preparation method and application thereof. The structural formula of the camellia oleifera shell polysaccharide is # imgabs0. The camellia oleifera shell polysaccharide of some examples is low in molecular weight, better in thermal stability, good in anti-inflammatory activity and free of toxicity to cells.

Description

Technical Field

[0001] The present invention belongs to the field of natural products, and particularly relates to a polysaccharide from Camellia oleifera fruit shell, a preparation method thereof and an application thereof. Background Art

[0002] Camellia oleifera Abel. is an evergreen shrub or small tree of the Theaceae family, and is a unique oil tree species in China, which is widely planted to produce high-quality edible oil. Camellia oleifera oil is rich in oleic acid and linoleic acid, has high nutritional value, and is deeply favored by the market. By-products - Camellia oleifera fruit shells are produced during the production process of Camellia oleifera oil. These fruit shells are usually discarded, piled up in fields or woodlands, or even randomly discarded, resulting in environmental pollution. In some cases, the fruit shells are burned, which not only wastes valuable resources but also causes serious harm to the atmospheric environment. With the in-depth research, people gradually realize that Camellia oleifera fruit shells are not useless. A large number of studies have shown that these by-products are rich in various bioactive substances with potential health benefits.

[0003] The oil-tea camellia fruit shell is mainly composed of lignin, cellulose, hemicellulose, polysaccharides, flavonoids, phenols and terpenoids. The research on oil-tea camellia fruit shell polysaccharides mainly focuses on the extraction of polysaccharides and process optimization. CN115448992A discloses a method for extracting oil-tea camellia fruit shell polysaccharides, which obtains oil-tea camellia fruit shell polysaccharides through steps such as crushing with a flash extractor, sieving, heating and extracting with distilled water, decompression concentration, precipitation with organic solvents, decolorization with activated carbon, and freeze-drying. CN105820263A discloses an extraction process for oil-tea camellia fruit shell polysaccharides, which includes the following steps: passing the oil-tea camellia fruit shell powder through a 40-60 mesh sieve to obtain treated powder; mixing the treated powder with water, where the mass ratio of the oil-tea camellia fruit shell powder to water is 1:10-20 to obtain a mixture; performing ultrasonic and / or microwave extraction on the mixture, where the conditions for ultrasonic extraction are: ultrasonic extraction at 80-100 °C for 15-35 min, and the conditions for microwave extraction are: maintaining at 240-700 W of microwave for 5-9 min; concentrating the extract at 40-50 °C and then adding ethanol, where the volume fraction of ethanol reaches more than 80%, and then performing alcohol precipitation at 3-5 °C for more than 12 hours, separating the precipitate to obtain the oil-tea camellia fruit shell polysaccharides. Shen Jianfu, Kang Haiquan, Chen Yaqi, etc. Research on the extraction and antioxidant effect of oil-tea camellia fruit shell polysaccharides [J]. Journal of the Chinese Cereals and Oils Association, 2010(8):4. Using the oil-tea camellia fruit shell as the raw material and taking the polysaccharide yield as the investigation index, based on the results of single-factor experiments, through orthogonal experiments, the effects of four factors, namely extraction time, extraction temperature, solid-liquid ratio, and ethanol concentration, on the polysaccharide yield of oil-tea camellia fruit shell were studied; and the antioxidant ability of oil-tea camellia fruit shell polysaccharides was evaluated by the TEAC method and the lipid antioxidant system. The results show that the extraction temperature has a significant effect on the polysaccharide yield of oil-tea camellia fruit shell, while the extraction time, solid-liquid ratio, and ethanol volume fraction have no significant effect on the polysaccharide yield of oil-tea camellia fruit shell. The optimal extraction process conditions for oil-tea camellia fruit shell polysaccharides are: temperature 90 °C, time 1.0 h, solid-liquid ratio 1:15, and ethanol volume fraction 80%. Under these conditions, the polysaccharide yield of oil-tea camellia fruit shell is 5.93%. Zhu Zhidong, Cai Yanqu, Lv Li, etc. Effects of different extraction methods on the polysaccharide content of oil-tea camellia fruit shell [J]. Journal of Guangzhou University of Chinese Medicine, 2019(3):4. Using the water extraction and alcohol precipitation method, alcohol-water extraction method, and ultrasonic-ethanol extraction method to extract oil-tea camellia fruit shell polysaccharides, and determining its content by the phenol-sulfuric acid method combined with ultraviolet-visible spectrophotometry.

Results

Conclusion

[0004] The purity of the oil-tea camellia fruit shell polysaccharides obtained by the existing methods is relatively low, and it is a mixture of various polysaccharides, which needs to be further optimized. Summary of the Invention

[0005] An object of the present invention is to overcome at least one deficiency of the prior art, and to provide a polysaccharide from camellia oleifera fruit shell, a preparation method thereof and an application thereof.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided:

[0008] A polysaccharide from camellia oleifera fruit shell, the main chain of which is →3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3)-α-D-Galp-(1→. The branched chains are α-D-Galp-(1→6)-α-D-Galp-(1→, α-L-Araf-(1→[5)-α-L-Araf-(1]7→3,5)-α-L-Araf-(1→, and α-L-Araf-(1→[4)-α-D-Glcp-(1]2→ and →3,6)-α-D-Galp-(1→, and its structural formula is as Figure 17 shown.

[0009] In the second aspect of the present invention, there is provided:

[0010] A preparation method of the polysaccharide from camellia oleifera fruit shell described in the first aspect of the present invention, comprising the following steps:

[0011] S1) Crushing and drying the camellia oleifera fruit shell; S2) Mixing the camellia oleifera shell powder with distilled water, performing water extraction at 70-90 °C, performing solid-liquid separation and concentration to obtain a concentrated solution; S3) Adding an ammonium sulfate solution and tert-butanol to the concentrated solution, oscillating sufficiently at 30-50 °C and then standing still to form a clear three-phase; S4) Collecting the lower layer solution in the three-phase, dialyzing using a dialysis membrane with a cut-off molecular weight of 3500 Da, and obtaining a crude polysaccharide after dialysis is completed; S5) Purifying the crude polysaccharide by DEAE-52 anion exchange column chromatography, collecting the chromatographic solution, concentrating, dialyzing using a dialysis membrane with a cut-off molecular weight of 3500 Da, and drying to obtain the polysaccharide from camellia oleifera fruit shell.

[0012] In some examples, the time for water extraction is 1-3 hours.

[0013] In some examples, the temperature for water extraction is 78-82 °C, and the extraction time is 110 min-130 min.

[0014] In some examples, the concentration of the ammonium sulfate solution is 20 w / v%, and the volume ratio of the ammonium sulfate solution to tert-butanol is 1:1.

[0015] In some examples, the dialysis time of the lower layer solution is 24-72 hours.

[0016] In some examples, when purifying crude polysaccharide, the concentration of the fed crude polysaccharide is adjusted to 18 - 24 mg / mL.

[0017] The above features can be arbitrarily combined without conflict.

[0018] The third aspect of the present invention provides:

[0019] Use of the polysaccharide from Camellia oleifera fruit shell described in the first aspect of the present invention in the preparation of an anti-inflammatory drug or dressing.

[0020] In some examples, the anti-inflammatory drug is used to treat colitis.

[0021] The fourth aspect of the present invention provides:

[0022] An anti-inflammatory composition added with the polysaccharide from Camellia oleifera fruit shell described in the first aspect of the present invention.

[0023] The beneficial effects of the present invention are:

[0024] The polysaccharide from Camellia oleifera fruit shell in some examples of the present invention has a lower molecular weight, better thermal stability, good anti-inflammatory activity, and no toxicity to cells.

[0025] The method for extracting the polysaccharide from Camellia oleifera fruit shell in some examples of the present invention is simple to operate and can prepare high-purity polysaccharide from Camellia oleifera fruit shell. Description of the Drawings

[0026] Figure 1 is the elution diagram of the polysaccharide from Camellia oleifera fruit shell on a DEAE-52 anion exchange column.

[0027] Figure 2 is the molecular weight characteristic map of the fructose CFP-A from Camellia oleifera fruit shell.

[0028] Figure 3 is the infrared spectrum of the polysaccharide CFP-A from Camellia oleifera fruit shell.

[0029] Figure 4 is the Congo red data of the polysaccharide CFP-A from Camellia oleifera fruit shell.

[0030] Figure 5 is the scanning electron microscope photograph of the polysaccharide CFP-A from Camellia oleifera fruit shell.

[0031] Figure 6 is the TG and DTG test curves of the polysaccharide CFP-A from Camellia oleifera fruit shell.

[0032] Figure 7 is the 1 1H NMR spectrum of the polysaccharide CFP-A from Camellia oleifera fruit shell.

[0033] Figure 8 is the13 13C NMR spectrum.

[0034] Figure 9 It is the DEPT-135 spectrum of camellia oleifera fruit shell polysaccharide CFP-A.

[0035] Figure 10 It is the COSY spectrum of camellia oleifera fruit shell polysaccharide CFP-A.

[0036] Figure 11 It is the HSQC spectrum of camellia oleifera fruit shell polysaccharide CFP-A.

[0037] Figure 12 It is the HMBC spectrum of camellia oleifera fruit shell polysaccharide CFP-A.

[0038] Figure 13 It is the NOESY spectrum of camellia oleifera fruit shell polysaccharide CFP-A.

[0039] Figure 14 It is the result of the anti-inflammatory experiment of camellia oleifera fruit shell polysaccharide CFP-A on cells.

[0040] Figure 15 and Figure 16 It is the inhibitory result of camellia oleifera fruit shell polysaccharide CFP-A on murine colitis.

[0041] Figure 17 It is the structural formula of camellia oleifera fruit shell polysaccharide CFP-A. Specific implementation mode

[0042] The technical solution of the present invention will be further described below in combination with examples and experiments.

[0043] Extraction of camellia oleifera fruit shell polysaccharide CFP-A:

[0044] S1) Crush the oil-tea camellia shell, sieve it through a 40-mesh sieve, and dry it under vacuum; S2) Mix the pretreated oil-tea camellia shell powder with distilled water at a ratio of 1:30 (g / mL), and boil it in a water bath at 80 °C for 2 hours; S3) Centrifuge twice at 4000 rpm for 10 minutes to collect and combine the supernatant; S4) Concentrate the combined supernatant to 1 / 3 of the original volume, add ammonium sulfate (20%, w / v) and tert-butanol (volume ratio of solution to tert-butanol: 1:1), and oscillate in a constant-temperature shaking water bath at 40 °C for 40 min. After oscillation, centrifuge at 4000 rpm for 10 min to form a clear three-phase; S5) Use a syringe to collect the lower layer of the solution (ammonium sulfate salt solution and polysaccharide), record the volume of the lower layer, and dialyze it with distilled water for 48 hours (MW: 3500 Da). During the dialysis process, change the dialysis pure water multiple times to maintain a high dialysis efficiency; S6) Dispense the dialyzed polysaccharide solution into disposable petri dishes, wrap it with aluminum foil, place it in -80 °C for 8 hours, and then put it into a freeze dryer for freeze-drying to obtain crude polysaccharide powder; S7) Dissolve the collected crude polysaccharide powder with deionized water to prepare a concentration of 20 mg / mL to obtain a crude polysaccharide solution. Filter the crude polysaccharide solution with a 40 μM microporous filter membrane to obtain a filtrate. Purify the crude polysaccharide solution (filtrate) by DEAE-52 anion exchange column chromatography, and collect the eluted chromatography solution; S8) Concentrate the collected chromatography solution with a rotary evaporator. After the concentrated solution cools, dispense it into a dialysis bag with a molecular weight cut-off of 3500 Da and dialyze it for 24 hours; S9) Dispense the collected purified polysaccharide solution into disposable petri dishes, package it completely, and place it in a -80 °C refrigerator for 8 hours to obtain purified oil-tea camellia shell polysaccharide CFP-A.

[0045] The elution diagram of oil-tea camellia shell polysaccharide on DEAE-52 anion exchange column is as Figure 1 shown. It can be seen from the figure that after purifying C-TPP using DEAE-52 anion exchange chromatography, four elution peaks were observed, named CFP-A, CFP-B, CFP-C, and CFP-D respectively. The yields of each component were 1.81%, 2.24%, 1.77%, and 2.76% respectively.

[0046] Characterization of oil-tea camellia shell polysaccharide CFP-A

[0047] Molecular weight characteristics of oil-tea camellia shell fructose

[0048] The molecular weight of CFP-A was determined by HPGPC method, and its molecular weight characteristics are as Figure 2As shown, calculated according to weight-average molecular weight (Mw) = -0.1973 RT + 11.738 (R² = 0.9956) and number-average molecular weight = -0.1964 RT + 11.7 (R² = 0.9955), the weight-average molecular weight (Mw) and number-average molecular weight of CFP-A are 5643 Da and 5622 Da. The polydispersity index of Mw / Mn is 1.00 (retained to 2 decimal places), indicating a high level of uniformity.

[0049] Infrared characteristics of Camellia oleifera fruit shell polysaccharide CFP-A

[0050] The FT-IR spectrum of CFP-A is as Figure 3 shown. CFP-A shows characteristic polysaccharide absorption peaks between 4000 and 400 cm -1 ⁻¹. The peak at 3400 cm -1 ⁻¹ is the stretching vibration of O-H, and the peak at 2936 cm -1 ⁻¹ is the stretching vibration of C-H. The peak at 1400 - 1200 cm -1 ⁻¹ is the characteristic absorption of the C-H band. The absorption characteristics near the peak at 1642 cm -1 ⁻¹ are due to the symmetric stretching of free carboxyl groups. The peak top at 1224 cm -1 ⁻¹ corresponds to the stretching vibration of C-O; the absorption peak at 1031 cm -1 ⁻¹ is due to the C-O-H stretching of glycosidic bonds. The absorption peak at 824 cm -1 ⁻¹ indicates the presence of α-type glycosidic bonds in CFP-A. In the UV-Vis spectrum, 280 cm -1 and 260 cm -1 are relatively smooth without absorption peaks, indicating that CFP-A lacks proteins or nucleic acids.

[0051] Congo red data of Camellia oleifera fruit shell polysaccharide CFP-A

[0052] To determine whether the polysaccharide chains in aqueous solution have a random coil structure or a triple helix conformation. Congo red can bind to the triple helix structure of polysaccharides to form a complex, resulting in a specific red shift in the maximum absorption wavelength (λmax) of the solution. The maximum absorption wavelengths of Congo red and CFP-A in NaOH solutions with different concentrations (0 - 0.4 M) are as Figure 4 shown. CFP-a does not show a triple helix shape, and compared with the Congo red solution, with the increase of NaOH concentration, CFP-a also does not show an obvious red shift or a substantial change in the maximum absorption wavelength.

[0053] Scanning electron microscope photos of Camellia oleifera fruit shell polysaccharide CFP-A are as Figure 5As shown, it can be seen that the surface of CFP-A is disordered, with many large and smooth pores of large diameter. The relatively weak intramolecular hydrogen bonds may be the reason for the loose and disordered surface of CFP-A, which is consistent with its relatively low molecular weight.

[0054] Thermodynamic Characteristics of Camellia oleifera Fruit Shell Polysaccharide CFP-A

[0055] The thermal stability of polysaccharides is usually evaluated using TG (thermogravimetry) and DTG (derivative thermogravimetry). The TG and DTG test curves depict two stages of weight loss of CFP-A ( Figure 6 ). In the initial weight loss stage (30°C - 222.02°C) of CFP-A, the weight loss rate is about 17.44%, indicating the evaporation of water in CFP-A, as it is known that polysaccharide samples contain some water. In the second weight loss stage at 222.02°C - 355.88°C, the weight loss is about 53.42%. Due to the depolymerization of the polysaccharide, the side chains may have broken and dehydrated. In addition, decarbonylation and decarboxylation may start at this step, resulting in weight loss. In the third weight loss stage at 355.88°C - 502.70°C, the weight loss is about 6.83%, and the weight loss is not obvious. In this stage, organic substances may be oxidized and decomposed. The DTG curve shows that CFP-A has the maximum weight loss at 292.64°C. Generally speaking, the smaller the molecular weight of a polysaccharide, the lower its thermal stability.

[0056] Structural Analysis of Camellia oleifera Fruit Shell Polysaccharide CFP-A

[0057] Figure 7 is the 1 1H NMR spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 8 is the 13 13C NMR spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 9 is the DEPT-135 spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 10 is the COSY spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 11 is the HSQC spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 12 is the HMBC spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. Figure 13 is the NOESY spectrum of Camellia oleifera fruit shell polysaccharide CFP-A. The chemical shifts of Camellia oleifera fruit shell polysaccharide CFP-A obtained from the above spectral data are shown in Table 1.

[0058] Table 1. Analysis Results of Chemical Shifts of Camellia oleifera Fruit Shell Polysaccharide CFP-A

[0059]

[0060] Analysis shows that the main chain of the polysaccharide CFP-A from Camellia oleifera fruit shell consists of →3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3)-α-D-Galp-(1→. The branched chains are α-D-Galp-(1→6)-α-D-Galp-(1→, α-L-Araf-(1→[5)-α-L-Araf-(1]7→3,5)-α-L-Araf-(1→, and α-L-Araf-(1→[4)-α-D-Glcp-(1]2→ as well as →3,6)-α-D-Galp-(1→. The structural formula is as follows:

[0061] .

[0062] Anti-inflammatory activity of polysaccharide CFP-A from Camellia oleifera fruit shell

[0063] RAW264.7 cells were cultured in a humid environment at 37 °C and 5% CO2. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. When the cells reached approximately 80% confluence, they were passaged.

[0064] RAW264.7 cells were seeded into 12-well plates at a density of 3×10 5 cells per well and incubated overnight in a humidified environment at 37 °C and 5% CO2. The experimental groups included an LPS-stimulated group (1 μg / mL), a positive control group (DEX, 1 μM), a blank control group (without LPS stimulation), and treatment groups (CFP-A: 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL). The cells were pretreated with the polysaccharide or DEX for 2 hours and then stimulated with 1 μg / mL LPS for 4 hours (RT-qPCR) or 24 hours (ELISA). This study investigated the anti-inflammatory activity of CFP-A based on an LPS-induced RAW264.7 cell inflammation model. The experimental results are as Figure 14 shown. CFP-A significantly downregulated the expression of IL-1β and IL-6 (p < 0.05) (Figures b, c). In addition, CFP-A had no toxicity to the cells.

[0065] Treatment of murine colitis with polysaccharide CFP-A from Camellia oleifera fruit shell

[0066] To induce colitis, 2.5% dextran sulfate sodium (DSS) was added to the drinking water for seven consecutive days. The results are as Figure 15 and Figure 16 shown. As Figure 15As shown in A. Compared with the control group, DSS administration led to weight loss (starting from day 4), accompanied by diarrhea and bloody stools. In addition, the disease activity index (DAI) increased (starting from day 4), the colon length shortened, and the spleen index increased. These symptoms were consistent with the previously established colitis model, successfully verifying the effectiveness of the acute colitis model. Based on this, we further investigated the improvement effects of 5-aminosalicylic acid (5-ASA) and different doses of CFP-A on these symptoms. Compared with the model group, treatment with 5-ASA and different doses of CFP-A led to weight recovery ( Figure 15 B), a decrease in the DAI level ( Figure 15 C), and an increase in the colon length ( Figure 15 E, Figure 16 G). The colon length in the H-CFP group was close to that of the control group, which directly reflected the improvement of colon inflammation. The spleen is a key lymphoid organ closely related to immune function. It is the largest immune organ in the body, and colitis can cause an increase in the spleen volume. The spleen volume and spleen index in the 5-ASA group and the CFP-A group were lower than those in the model group, indicating that CFP-A slowed down the decrease in the spleen index of mice ( Figure 15 D, Figure 16 F). Overall, CFP-A alleviated the clinical symptoms of colitis mice. This indicates that CFP-A has a protective effect on DSS-induced colon inflammation.

[0067] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. An oil-tea camellia shell polysaccharide, whose main chain consists of →3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3,6)-α-D-Galp-(1→3)-α-D-Galp-(1→. The branched chains are α-D-Galp-(1→6)-α-D-Galp-(1→, α-L-Araf-(1→[5)-α-L-Araf-(1]7→3,5)-α-L-Araf-(1→, and α-L-Araf-(1→[4)-α-D-Glcp-(1]2→ as well as →3,6)-α-D-Galp-(1→, and its structural formula is shown in Figure 17.

2. The preparation method of the oil-tea camellia shell polysaccharide according to claim 1, comprising the following steps: S1) Crush and dry the oil-tea camellia shells; S2) Mix the oil-tea camellia shell powder with distilled water, extract with water at 70-90 °C, perform solid-liquid separation and concentration to obtain a concentrated solution; S3) Add an ammonium sulfate solution and tert-butanol to the concentrated solution, shake well at 30-50 °C and then let it stand to form a clear three-phase; S4) Collect the lower-layer solution in the three-phase, dialyze it using a dialysis membrane with a molecular weight cut-off of 3500 Da, and obtain a crude polysaccharide after dialysis; S5) Purify the crude polysaccharide by DEAE-52 anion exchange column chromatography, collect the chromatography solution, concentrate it, dialyze it using a dialysis membrane with a molecular weight cut-off of 3500 Da, and dry it to obtain the oil-tea camellia shell polysaccharide.

3. The method according to claim 2, wherein The time for water extraction is 1-3 hours.

4. The method according to claim 2, wherein The temperature for water extraction is 78-82 °C, and the extraction time is 110 min-130 min.

5. The method according to claim 2, wherein The concentration of the ammonium sulfate solution is 20 w / v%, and the volume ratio of the ammonium sulfate solution to tert-butanol is 1:

1.

6. The method according to claim 2, characterized in that, The dialysis time of the lower-layer solution is 24-72 hours.

7. The method according to claim 2, characterized in that, When purifying the crude polysaccharide, the concentration of the crude polysaccharide in the inlet liquid is adjusted to 18-24 mg / mL.

8. The application of the oil-tea camellia shell polysaccharide according to claim 1 in the preparation of anti-inflammatory drugs or dressings.

9. The application according to claim 8, wherein The anti-inflammatory drug is used for treating colitis.

10. An anti-inflammatory composition, characterized in that, It is added with the oil-tea camellia shell polysaccharide according to claim 1.

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