A method for extracting oil tea polysaccharides based on deep eutectic solvent and its application

Abstract

The present invention discloses a method for extracting camellia oleifera polysaccharides based on a deep eutectic solvent and its application. The present invention uses a deep eutectic solvent for the extraction of camellia oleifera polysaccharides for the first time and screens out the most suitable deep eutectic solvent system Bet-LA (1:3) for the extraction of camellia oleifera polysaccharides. The extraction rate is 2.5 times higher than that of the traditional water extraction method. In addition, the present invention demonstrates the antagonistic heavy metal effect of camellia oleifera polysaccharides from the physiological and biochemical levels of Caenorhabditis elegans, providing a theoretical basis and theoretical basis for the development of camellia oleifera polysaccharides into a natural medicine for treating heavy metal poisoning.

Description

Technical Field

[0001] The present invention belongs to the field of biological extraction technology, and in particular relates to a method for extracting tea leaf polysaccharides based on a low eutectic solvent and an application thereof. Background Art

[0002] Camellia oleifera Abel is an evergreen small tree in the genus Camellia, Theaceae family. Its main economic value lies in the production of tea oil. The area of camellia oil plantations in my country is expected to reach 6.003 million hectares by 2025. 2 To increase tea oil leaf yield, annual pruning of seedlings results in the discarding of one-third of the leaves from the entire plant, resulting in a significant waste of resources. Tea oil leaves contain a variety of bioactive components, primarily polysaccharides, polyphenols, and saponins, which have antioxidant, anti-tumor, hypoglycemic, and antibacterial properties. Therefore, studying the utilization value of tea oil leaves is of great practical significance for improving their comprehensive utilization rate.

[0003] Polysaccharides are extremely important active ingredients in Camellia oleifera leaves and have gradually attracted attention due to their antioxidant, anti-tumor and hypoglycemic effects. Currently, the main extraction methods for C. oleifara leaves polysaccharides (CLPs) are water extraction methods (e.g., GC-MS analysis, ultrasound-assisted hot water extraction, hot water extraction, etc.). However, traditional water extraction methods have disadvantages such as low extraction yield and weak extract activity. Therefore, the field urgently needs to develop a new green and efficient extraction solvent for the extraction of C. oleifara leaves polysaccharides to improve their yield and further expand their application in food, medicine and other fields.

[0004] Deep eutectic solvents (DESs) are a class of green, biodegradable solvents. DESs are primarily composed of two or more hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) that are perfectly combined through hydrogen bonding. In recent years, DESs have been used for the extraction of bioactive compounds due to their high efficiency, environmental friendliness, and low cost. Therefore, identifying a DES solvent suitable for extracting Camellia oleifera polysaccharides and optimizing extraction conditions are crucial for addressing the low extraction yields of traditional water extraction methods and improving the overall utilization of Camellia oleifera. Summary of the Invention

[0005] In view of this, in order to overcome the technical problem of low extraction rate of polysaccharides by traditional water extraction method, the present invention uses oil tea as raw material, utilizes the screened DES-3 to extract oil tea polysaccharides and optimizes the extraction method, and provides a method for extracting oil tea polysaccharides based on low eutectic solvent in the field. The method has the advantages of high extraction rate, good extract activity, green environmental protection, simple operation, etc.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] A first aspect of the present invention provides a method for extracting tea leaf polysaccharides based on a deep eutectic solvent.

[0008] Furthermore, the method comprises the following steps:

[0009] (1) drying and crushing the oil tea leaves to obtain oil tea leaf powder;

[0010] (2) extracting the camellia oleifera powder described in step (1) using a deep eutectic solvent Bet-LA to obtain a camellia oleifera polysaccharide extract;

[0011] (3) subjecting the oil tea polysaccharide extract described in step (2) to alcohol precipitation, collecting the precipitate, and freeze-drying to obtain oil tea polysaccharide;

[0012] In the deep eutectic solvent Bet-LA, the hydrogen bond acceptor is betaine and the hydrogen bond donor is lactic acid.

[0013] Furthermore, the molar ratio of betaine to lactic acid in the deep eutectic solvent Bet-LA is 1:(1-10);

[0014] Preferably, the molar ratio of betaine to lactic acid in the deep eutectic solvent Bet-LA is 1:3.

[0015] Furthermore, the water content of the deep eutectic solvent Bet-LA is 10%-50%;

[0016] Preferably, the water content of the deep eutectic solvent Bet-LA is 30%.

[0017] In the present invention, Bet in the deep eutectic solvent Bet-LA refers to betaine (Betaine, Bet), and LA in the deep eutectic solvent Bet-LA refers to lactic acid (Lactic acid, LA). The reagents are all conventional reagents in the art and can be obtained through conventional purchasing channels.

[0018] In some embodiments, the low eutectic solvent Bet-LA is prepared by the following preparation method: the above-mentioned betaine and lactic acid are placed in a beaker according to a certain molar ratio, heated and stirred until a transparent and uniform liquid is formed, taken out and naturally cooled to room temperature. If no precipitate is formed, it indicates that the low eutectic solvent Bet-LA is successfully synthesized.

[0019] In the present invention, the inventors verified through a large number of comparative experiments that not all conventional low eutectic solvents can be used for the effective extraction of tea leaf polysaccharides. Specifically, the present invention measured and compared the polysaccharide extraction effects and physicochemical properties of 11 different types of low eutectic solvents (Bet-U, Bet-MeU, Bet-Gly, Bet-PG, Bet-EG, Bet-LeA, Bet-LA, Bet-BG, Bet-Sorb, Bet-MA, Bet-Xyl). The results showed that among the 11 different types of low eutectic solvents, only 4 low eutectic solvents had higher polysaccharide extraction rates than the traditional water extraction method. Among them, Bet-LA adopted in the present invention had the highest extraction rate. This technical effect is a technical effect that is unexpected by those skilled in the art based on the content disclosed in the prior art.

[0020] In the present invention, the betaine and lactic acid are both natural organic compounds, which can form a uniform deep eutectic solvent by heating and stirring. By adding an appropriate amount of water to the deep eutectic solvent, the viscosity of the deep eutectic solvent can be reduced, which is beneficial to the diffusion of the active ingredients of the extract and the penetration of solvent protons during extraction.

[0021] Furthermore, the liquid-to-solid ratio of the low eutectic solvent Bet-LA and the oil tea leaf powder in step (2) is (5-25) mL / g; preferably, the liquid-to-solid ratio of the low eutectic solvent Bet-LA and the oil tea leaf powder in step (2) is 14.4 mL / g.

[0022] Furthermore, the extraction temperature in step (2) is (60-80)°C;

[0023] Preferably, the extraction temperature in step (2) is 71°C;

[0024] Preferably, the extraction time in step (2) is (60-80) min;

[0025] More preferably, the extraction time in step (2) is 69 min;

[0026] Preferably, the extraction method in step (2) is constant temperature water bath extraction.

[0027] In some embodiments, the specific experimental method of the constant temperature water bath extraction is as follows: weigh the oil tea leaf powder, add the low eutectic solvent Bet-LA according to a certain liquid-to-solid ratio, extract for a certain time in a constant temperature water bath at a certain temperature, filter the obtained supernatant, and obtain the oil tea leaf polysaccharide extract after centrifugation.

[0028] Furthermore, the alcohol precipitation in step (3) is carried out using ethanol.

[0029] Further, the alcohol precipitation comprises the following steps:

[0030] ① After concentrating the oil tea leaf polysaccharide extract by rotary evaporation, ethanol was added to obtain a precipitate;

[0031] ② Add Sevag reagent to the precipitate to remove protein and collect the supernatant;

[0032] ③ dialyzing the supernatant to remove the deep eutectic solvent Bet-LA, concentrating and freeze-drying to obtain Camellia oleifera polysaccharide;

[0033] Preferably, the Sevag reagent is a reagent comprising n-butanol and chloroform;

[0034] Preferably, the ratio of n-butanol to chloroform in the Sevag reagent is 1:4.

[0035] Furthermore, the ethanol is anhydrous ethanol.

[0036] Furthermore, the added volume of the anhydrous ethanol is 9 times the volume of the supernatant of the oil tea leaf polysaccharide after rotary evaporation concentration.

[0037] In some embodiments, the specific experimental method of the alcohol precipitation is as follows: the tea polysaccharide extract obtained in step (2) is filtered and centrifuged, and the supernatant is collected. After removing excess water by rotary evaporation, 9 volumes of anhydrous ethanol are added, and the mixture is kept at 4°C overnight. The precipitate is collected by centrifugation, and the precipitate is dissolved in water and Sevag reagent (n-butanol: chloroform = 1:4) is added to repeatedly remove protein until no protein precipitates. The supernatant is collected and dialyzed for about 3 days, with the water changed 3 to 5 times a day, to remove the low eutectic solvent as much as possible. The dialyzed liquid is concentrated and freeze-dried to obtain oil tea polysaccharide.

[0038] The present invention also provides a method for recovering the deep eutectic solvent Bet-LA described in the first aspect of the present invention.

[0039] Furthermore, the method includes the following steps: collecting the sample effluent, collecting the supernatant containing the low eutectic solvent Bet-LA after alcohol precipitation, and removing the water and ethanol therein by rotary evaporation, which can be reused for the extraction of oil tea polysaccharides, thereby completing the recovery of the low eutectic solvent Bet-LA.

[0040] In some embodiments, the recovered low eutectic solvent Bet-LA is subjected to rotary evaporation to remove excess water and ethanol, and the oil tea polysaccharides are re-extracted according to the steps described in the first aspect of the present invention until the extraction rate of oil tea polysaccharides is less than 80%, which means that the extraction efficiency has dropped significantly and cannot be reused.

[0041] In a specific embodiment of the present invention, the present invention has experimentally verified that as the number of recycling times increases, the low eutectic solvent Bet-LA provided by the present invention still has a large extraction capacity, and the extraction efficiency of Camellia oleifera polysaccharides using the low eutectic solvent Bet-LA recovered for the third time can still be as high as 87.1%.

[0042] The second aspect of the present invention provides oil tea polysaccharide prepared by the method described in the first aspect of the present invention.

[0043] The third aspect of the present invention provides any of the following applications:

[0044] (1) Use of the oil tea polysaccharide according to the second aspect of the present invention in the preparation of a drug for alleviating heavy metal toxicity or treating heavy metal poisoning;

[0045] (2) Use of the oil tea polysaccharide according to the second aspect of the present invention in the preparation of antioxidant products and / or blood sugar-lowering products;

[0046] (3) Application of the low eutectic solvent Bet-LA described in the first aspect of the present invention in the extraction of Camellia oleifera polysaccharides.

[0047] Furthermore, the heavy metals include Al, Hg, Pb, Cd, Cr and / or Ni.

[0048] The present invention also provides the use of tea leaf polysaccharide in preparing anti-tumor products and / or antibacterial products.

[0049] In some embodiments, the product includes but is not limited to: a drug, a pharmaceutical composition or a pharmaceutical preparation, the main active ingredient of which is the oil tea leaf polysaccharide prepared by the method according to the first aspect of the present invention.

[0050] In some embodiments, the dosage form of the drug, pharmaceutical composition or pharmaceutical preparation includes a dosage form for administration through the gastrointestinal tract and a dosage form for parenteral administration. In some embodiments, the dosage form for administration through the gastrointestinal tract includes a solution, a granule, a tablet, a capsule, a suspension, a powder, a sustained-release agent, an effervescent agent, an emulsion, a syrup, a drop, or a chewable agent. In some embodiments, the dosage form for parenteral administration includes an injection dosage form, a respiratory tract dosage form, a cavity dosage form, a mucosal dosage form, or a skin dosage form.

[0051] In some embodiments, the appropriate dosage of the drug, pharmaceutical composition or pharmaceutical preparation can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity of the subject. A skilled physician can usually easily determine the prescription and the desired dosage that is effective for treatment.

[0052] In a specific embodiment of the present invention, using Caenorhabditis elegans, the present invention demonstrates that tea leaf polysaccharides prepared using the method described in the first aspect of the present invention enhance the nematode's resistance to heavy metal stress by regulating the nuclear import of the nematode DAF-16, SKN-1, and HSF-1 nuclear transcription factors, thereby activating the nematode's oxidative defense system, reducing ROS and MDA levels in the nematode, reducing lipid and lipofuscin accumulation, and alleviating intestinal damage. The research results confirm that tea leaf polysaccharides have the effect of alleviating the toxicity of various heavy metals. This research achievement provides a theoretical basis and theoretical foundation for the development of tea leaf polysaccharides as a natural medicine for treating heavy metal poisoning.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] (1) The present invention is the first to use a deep eutectic solvent for the extraction of Camellia oleifera polysaccharides and screen out the most suitable deep eutectic solvent system Bet-LA (1:3) for the extraction of Camellia oleifera polysaccharides. The extraction rate is 2.5 times higher than that of the traditional water extraction method. In addition, the present invention demonstrates the antagonistic heavy metal effect of Camellia oleifera polysaccharides at the physiological and biochemical level of Caenorhabditis elegans, providing a theoretical basis and theoretical foundation for the development of Camellia oleifera polysaccharides into a natural medicine for the treatment of heavy metal poisoning.

[0055] (2) The present invention provides a new process technology for extracting camellia oil polysaccharides based on the low eutectic solvent Bet-LA in the technical field of camellia oil polysaccharide extraction, and realizes the reuse of the low eutectic solvent. It has the advantages of being green, environmentally friendly, efficient, reusable, simple to operate, high extraction rate, and good extract activity. It provides new ideas and strategies for the extraction of camellia oil polysaccharides and can realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : Effects of different DESs on the extraction rate of CLPs, (A) extraction rate of betaine-based DESs, (B) extraction rate of different molar ratios of Bet-LA.

[0057] Figure 2 : FTIR and 1 H NMR spectra, (A) FTIR spectra, (B) Bet and LA 1 H NMR spectra, (C) DESs with different molar ratios1 H NMR spectrum.

[0058] Figure 3 : DSC curves and physicochemical properties of DESs, (A) DSC curves of Bet and LA, (B) DSC curves of DESs with different molar ratios, (C) density and polarity of DESs with different molar ratios, (D) pH, conductivity and viscosity of DESs with different molar ratios.

[0059] Figure 4 : Evaluation of the recovery performance of DES-3.

[0060] Figure 5 :The effects of different factors on the extraction rate of CLPs, (A) the effect of liquid-to-solid ratio, (B) the effect of water content, (C) the effect of extraction time, and (D) the effect of extraction temperature.

[0061] Figure 6 : 3D response surface diagram of the interaction effect of various factors in DES extraction method on the extraction rate of oil tea polysaccharides, (A) extraction time, water content, (B) extraction temperature, water content, (C) extraction time, extraction temperature, (D) liquid-to-solid ratio, water content, (E) extraction time, liquid-to-solid ratio, (F) liquid-to-solid ratio, extraction temperature.

[0062] Figure 7 : 2D contour map of the interaction effect of various factors in DES extraction method on the extraction rate of oil tea polysaccharides, (A) extraction time, water content, (B) extraction temperature, water content, (C) extraction time, extraction temperature, (D) liquid-to-solid ratio, water content, (E) extraction time, liquid-to-solid ratio, (F) liquid-to-solid ratio, extraction temperature.

[0063] Figure 8 : Physicochemical characteristics of CLPs, (A) monosaccharide composition, (B) FTIR spectrum, (C) DSC curve, (D) TGA curve.

[0064] Figure 9 : Scanning electron micrographs, (A) CLP-W, (B) CLP-D, (C) oil-tea camellia powder, (D) residue after hot water extraction, (E) residue after DES-3 extraction.

[0065] Figure 10 : Emulsification ability of CLPs, (A) fresh emulsion, (B) emulsion after 30 days of storage, (C) water-oil mixture, (D) CLP-W emulsion, (E) micrographs of CLP-D emulsion, (F) emulsification stability and emulsification activity, (G) particle size and potential value.

[0066] Figure 11 : Effects of CLPs on the survival rate of Caenorhabditis elegans under Hg stress, (A) CLP-W treatment, (B) CLP-D treatment.

[0067] Figure 12 :Effects of CLPs on the survival rate of nematodes under different heavy metal stresses, (A) Al treatment, (B) Cr treatment, (C) Cd treatment, (D) Pb treatment, (E) Ni treatment, (F) Hg treatment. DETAILED DESCRIPTION

[0068] The present invention will be further described below with reference to specific embodiments. The specific embodiments are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and intent of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0069] The drugs, reagents, and raw materials used in the present invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained from commercial sources. Experimental methods not specifying specific conditions in the present invention are generally carried out under conventional conditions or conditions recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0070] Example 1 Analysis of the extraction rate and physicochemical properties of different DESs

[0071] 1. Experimental Materials

[0072] The oil tea leaves were collected from the oil tea planting base in Tianquan County, Ya'an City, Sichuan Province. The oil tea leaves were washed, dried, and crushed into powder for later use.

[0073] 2. Experimental methods

[0074] 2.1DESs solvent synthesis

[0075] The specific operation is as follows: Betaine (Bet) as HBA and 11 different HBDs including urea (Urea), methylurea (MeU), glycerol (Gly), 1,2-propylene glycol (PG), ethylene glycol (EG), 1,3-butylene glycol (BG), sorbitol (D-sorbitol, Sorb), xylitol (Xyl), levulinic acid (LeA), lactic acid (LA), and malic acid (MA) are weighed in a molar ratio of 1:1, placed in a 100mL beaker, wrapped with tin foil, heated and stirred until a transparent and uniform liquid is formed, and then taken out and naturally cooled to room temperature. If no precipitate is formed, the DESs are successfully synthesized.

[0076] 2.2 Determination of physical and chemical properties of DESs

[0077] The viscosity, conductivity, pH, and density of the prepared DESs were measured (using conventional methods known in the art).

[0078] 2.3DESs screening

[0079] Camellia oleifera powder was weighed and DES solvent was added at a solid-liquid ratio of 10 g / mL. Extraction was performed in a 65°C waterbath for 60 minutes. The supernatant was filtered and centrifuged to obtain the polysaccharide extract, with hot water extraction serving as a control. The extraction yield was determined using the phenol-sulfuric acid method: 10 μL of the crude extract was aspirated and mixed with UP water to a 1 mL solution. Finally, 200 μL of the dilution was transferred to a numbered EP tube, 0.5 mL of concentrated sulfuric acid and 0.1 mL of 6% phenol solution were added, and the mixture was shaken thoroughly. The mixture was reacted in a 90°C waterbath for 5 minutes and at room temperature for 20 minutes. The OD value was then measured at 490 nm. Glucose was used as the standard, and a standard curve was generated. The absorbance of the extract was substituted into the standard curve to calculate the extraction yield, thus identifying the DES with the best extraction yield.

[0080] 2.4 Screening of the optimal molar ratio of Bet-LA

[0081] Based on the extraction yields of different DESs, Bet-LA showed the best extraction efficiency. Therefore, we further prepared DESs (DES-1, DES-2, DES-3, DES-4, and DES-5) containing different Bet-LA molar ratios (1:1, 1:2, 1:3, 1:4, and 1:5), measured and compared their extraction yields, and finally screened for the optimal molar ratio.

[0082] Infrared spectroscopy analysis of 2.5DESs

[0083] Potassium bromide was dried and ground into powder using an agate mortar, and then pressed into transparent thin slices. Lactic acid monomer and Bet-LA with different molar ratios were applied to the slices using cotton swabs, and then placed in a Fourier transform infrared spectrometer at 400-4000 cm -1 The wavelength range was scanned, and the betaine monomer was mixed with potassium bromide powder at a ratio of 1:100 and ground into a sheet for scanning and measurement.

[0084] 2.6 Nuclear Magnetic Resonance Spectroscopy Determination of DESs

[0085] DESs and their monomers were dissolved in D2O and tested on a 400-MHz Bruker AVANCE NMR spectrometer (Bruker, Switzerland). 1 H NMR analysis and identification.

[0086] 2.7 Thermal stability determination

[0087] 2 mg of DESs and monomer samples were sealed in Al pans for DSC analysis. Under a nitrogen atmosphere, the pans were heated from 20°C to 400°C at a rate of 10°C / min. An empty pan was used as a control, and indium metal was used for calibration.

[0088] 2.8 Polarity determination

[0089] Polarity also has a significant impact on extraction efficiency and can be measured by fluorescence emission maxima. Using the Nile Red polarity scale as a solvatochromic probe, different DESs were mixed and the UV-visible absorption spectrum maxima were recorded. When Nile Red interacts with the solvent, polarity can be represented by the shift in λmax. ENR is calculated by the following formula: NR =28591 / λmax.

[0090] 2.9 Recycling and Reuse of DES-3

[0091] DESs can be recycled and reused during the extraction process, significantly reducing production costs. A recovery test was conducted using the DES-3 system, which exhibits the best extraction yield. The procedure was as follows: 20.21 g of DES-3 was weighed and the volume recorded. The volume was then filled into a 50 mL centrifuge tube, and 33% water was added to make up to 25 mL. The polysaccharide was extracted under optimal conditions. After alcohol precipitation, the supernatant containing DES-3 was collected. The water and ethanol were then removed using a rotary evaporator until no more liquid flowed out. The recovered liquid, W2, was weighed, and the DES-3 recovery was calculated as W2 / 20.21 g. The polysaccharide (E1) extraction yield was also determined using the absorbance of the recovered DES-3 as a blank. The recovered DES-3 was then subjected to secondary and tertiary extraction using the same process to obtain E2, E3, and W3. The extraction efficiency was expressed as the ratios of E2 / E1 and E3 / E1.

[0092] 3. Experimental results

[0093] 3.1 Screening of DESs

[0094] In order to screen the best extraction solvent, the extraction effect and physicochemical properties of polysaccharides from 11 different types of DESs were determined. The results are shown in Tables 1 and Figure 1 A, among which Bet-LA has a lower pH value and the highest extraction rate. Viscosity is another important characteristic of DESs in applications. In this study, Bet-Sorb had the highest viscosity but the lowest extraction rate, while other DESs with lower viscosities than Bet-Sorb had higher extraction rates than Bet-Sorb, which may be because the high viscosity solvent hinders mass transfer between the raw material and the extraction solvent. Compared with the traditional solvent water, most DESs have a higher binding affinity with polysaccharides due to hydrogen bonds and electrostatic interactions between different solvents, thus showing a higher extraction ability. However, in this study, only four DESs had a higher polysaccharide extraction rate than the water extraction method (7.61%), among which Bet-LA had the highest extraction rate (16.57%).

[0095] Table 1 Physicochemical properties of DESs

[0096]

[0097]

[0098] 3.2 Effect of Bet-LA molar ratio on extraction yield

[0099] DESs (DES-1, DES-2, DES-3, DES-4, DES-5) with different molar ratios of Bet-LA (1:1, 1:2, 1:3, 1:4, 1:5) were used to extract polysaccharides from Camellia oleifera. Figure 1As shown in Figure 2B, the extraction yield of DESs initially increased and then decreased with increasing Bet-LA molar ratios. When the Bet-LA molar ratio was 1:3, the extraction yield reached a maximum of 18.23%, but this gradually decreased with increasing molar ratios. This can be explained by the lower viscosity and strongest hydrogen bonding interactions of DESs at a Bet-LA molar ratio of 1:3, which facilitates solvent penetration and promotes the dissolution of Camellia oleifera polysaccharides. DES-3 was ultimately selected for subsequent experiments.

[0100] 3.3 FTIR analysis of DESs with different molar ratios of Bet-LA

[0101] The criterion for the success of DESs synthesis is the formation of hydrogen bonds. FTIR analysis can be used to evaluate whether hydrogen bonds are formed between HBAs and HBDs during DESs synthesis. Figure 2 As shown in A, in betaine monomer, the -1 and 1334.5cm -1 The absorption bands corresponding to the carbonyl group (C=O) and the quaternary ammonium structure (CN) were observed at 1736.5 cm-1. In addition, the FTIR spectrum of lactic acid showed that the stretching frequencies of its carboxyl group (-COOH) and hydroxyl group (-OH) were 1736.5 cm-1, ... -1 and 3493.2cm -1 In the tested DESs, the -OH band shifted to a lower absorption peak, indicating the formation of hydrogen bonds between betaine and lactic acid in the eutectic mixtures except for DES-1 and DES-2.

[0102] 3.4 Bet-LA DESs with different molar ratios 1 H NMR analysis

[0103] pass 1 H NMR analysis further confirmed the intermolecular interaction between betaine and lactic acid DESs. 1 H NMR spectrum Figure 2 As shown in BC. Obviously, there is no significant change in the chemical shift of -CH3 from betaine (3.27ppm) and lactic acid (1.41ppm) to the corresponding DESs. The same phenomenon exists for -OH in lactic acid (4.37ppm), indicating that the hydrogen bonds in DESs are formed by two carboxylic acids, rather than secondary hydroxyl and carboxylic acid groups. Since the chemical shift of active hydrogen atoms is imprecise, only -CH2 in betaine (3.91ppm) changes slightly to 3.96ppm downfield in DESs. In summary, the test samples in this study 1 H NMR spectroscopy provided evidence for the successful formation of hydrogen bonds between betaine and lactic acid.

[0104] DSC analysis of DESs with different molar ratios of 3.5Bet-LA

[0105] DSC is the main method to study the phase transition of DESs. Figure 3 As shown in Figures AB, lactic acid exhibits a sharp endothermic peak at 87.3°C, representing its phase transition temperature. Betaine exhibits an endothermic peak at 313.2°C, indicating its melting point. All DESs exhibit endothermic peaks after 100°C, demonstrating their excellent thermal stability and promising application in polysaccharide extraction.

[0106] 3.6 Analysis of physicochemical properties of Bet-LA at different molar ratios

[0107] The physicochemical properties of the solvent play an important role in the extraction efficiency. From the effect of the single factor molar ratio on the extraction rate, it can be seen that DES-3 has a better extraction ability for polysaccharides. This study measured the physicochemical properties of DESs with different moles and conducted a comparative analysis to further explore the factors that affect the extraction effect of DESs. Figure 3 As can be seen from the CD, the pH and viscosity of the DESs decreased significantly with the increase of the molar ratio of betaine to lactic acid. However, the opposite trend was observed in the conductivity. The density of the DES was the highest when the Bet-LA molar ratio was 1:3, which may be due to the strongest hydrogen bonding interaction between betaine and lactic acid. The relative polarity of the measured DESs was expressed as E NR According to the Nile Red polarity scale, high E NR Value represents low polarity, low E NR The value indicates the high polarity of the solvent. Figure 3 C It can be seen that the ENR value of DESs decreases with the increase of the molar ratio of lactic acid, which means that the polarity of DESs gradually increases with the increase of HBD content. In summary, the strong extraction ability of DES-3 may be related to the suitable physicochemical properties and strong hydrogen bonding interaction of Bet-LA DES at the optimal molar ratio of 1:3.

[0108] 3.7 Evaluation of the recovery performance of DES-3

[0109] DESs can be recycled after production, which is one of their advantages as green solvents. In this study, DES-3 was used as an example to evaluate its recycling performance. Figure 4 As shown in the figure, with the increase of the number of cycles, the extraction rate, extraction efficiency and recycling rate of DES-3 gradually decreased, but it still had a large extraction capacity. The extraction efficiency of camellia oleifera polysaccharides using the third recycled DES-3 was still up to 87.1%.

[0110] Example 2 Optimization of CLPs extraction

[0111] 1. Experimental methods

[0112] 1.1 Single-factor experiment

[0113] A single-factor experiment was conducted with the yield of oil tea polysaccharides as the evaluation index, and the effects of DESs water content (10%, 30%, 50%, 70% and 90%); liquid-to-solid ratio (5:1, 10:1, 20:1, 30:1, 40:1 and 50:1 mL / g); extraction temperature (55, 65, 75, 85, and 95°C) and extraction time (30, 45, 60, 75 and 90 min) on the yield of oil tea polysaccharides were investigated.

[0114] 1.2 Response surface analysis

[0115] Based on the results of the single-factor experiments, four independent variables were identified: extraction time, extraction temperature, water content, and liquid-to-solid ratio. Response surface analysis software, Box-Behnken, was used to design the experiment. Variance analysis was performed using polysaccharide yield as the response value. Ultimately, the optimal process conditions for tea polysaccharide extraction were determined. The experimental design is shown in Table 2.

[0116] Table 2 Factors and levels involved in BBD

[0117]

[0118] 1.3 Model Validation

[0119] Multivariate quadratic regression analysis was performed on the data from the optimized DES-3 extraction method for Camellia oleifera polysaccharides, revealing the optimal extraction conditions. Considering operational feasibility, the predicted conditions were slightly modified to: a liquid-to-solid ratio of 14.4 mL / g, an extraction temperature of 71°C, an extraction time of 69 minutes, and a water content of 30%. Camellia oleifera polysaccharides were extracted under these conditions, and the polysaccharide extraction yield was calculated. Each experiment was repeated three times, and the statistical differences between the measured values and the model-predicted values were analyzed.

[0120] 1.4 Preparation of CLPs

[0121] A large amount of Camellia oleifera powder was weighed and Camellia oleifera polysaccharides were extracted according to the optimized process conditions. After filtration and centrifugation, the supernatant was collected and concentrated by rotary evaporation to remove excess water. Nine volumes of anhydrous ethanol were added and the mixture was incubated at 4°C overnight. The precipitate was collected by centrifugation and dissolved in water. Sevag reagent (n-butanol: chloroform = 1:4) was added for repeated deproteinization until no protein precipitated. The supernatant was collected and dialyzed for approximately 3 days, with the water changed 3-5 times daily to remove as much DES solvent as possible. The dialyzed liquid was concentrated and freeze-dried to obtain CLP-D. Simultaneously, hot water extraction was used as a control to extract Camellia oleifera polysaccharides to obtain CLP-W.

[0122] 2. Experimental results

[0123] 2.1 Univariate analysis

[0124] Single factor test was conducted on the extraction conditions such as liquid-to-solid ratio, water content, extraction time, and extraction temperature. The results are as follows: Figure 5 In this study, we used DES-3 to investigate the effects of these factors on the extraction of polysaccharides from Camellia oleifera. Figure 5 A shows that within a certain liquid-to-solid ratio range, the polysaccharide extraction rate increases with the increase of the liquid-to-solid ratio, reaching a maximum value of 18.72% at 10:1 mL / g, and then decreasing gently. Generally, an increase in the liquid-to-solid ratio can promote the dispersion of the target extract in the solvent, thereby improving the extraction efficiency. However, if the liquid-to-solid ratio is too large, it will affect the subsequent treatment, resulting in a decrease in the extraction rate or gentle fluctuations. Therefore, this study set the liquid-to-solid ratio range of RSM to 5:1, 10:1 and 15:1 mL / g. In addition, from Figure 5 B shows that the extraction rate of DES-3 first increases and then decreases with the increase of the water content of DES-3. The extraction rate can reach a maximum of 18.34% at a water content of 30%. When the water content exceeds the optimal water content of 30%, the extraction rate decreases. This may be due to the changes in the polarity, pH value, viscosity and other properties of DESs after adding water. Adding water to DESs will weaken the hydrogen bonding between DESs and polysaccharides. Therefore, in order to clarify the optimal water content of DESs, the water content range used in the RSM analysis is 10%, 30% and 50%. In addition, Figure 5 The results of C show the change of extraction rate with extraction time. In the period of 30 to 75 minutes, as the extraction time increases, the extraction rate of tea oil leaf polysaccharides continues to increase, and the extraction rate reaches a maximum of 18.47% at 75 minutes. However, on the contrary, when the time is longer than 75 minutes, the extraction rate decreases, which may be because the polysaccharides have been fully extracted and long-term extraction may cause polysaccharide decomposition. In order to obtain clearer optimal extraction time conditions, extraction times of 45, 60 and 75 minutes were selected in the RSM design. Temperature is also a key factor in separating polysaccharides from raw materials. Figure 5 As shown in D, the extraction rate gradually increased from 55°C to 65°C to 18.31%, and then decreased significantly. In order to obtain the optimal temperature conditions, we selected the extraction temperature range of 55, 65 and 75°C for RSM analysis.

[0125] 2.2 Response surface design and analysis of variance

[0126] Based on the results of the single-factor experiment, a four-factor, three-level experimental design was subjected to RSM analysis. A total of 29 experimental groups were set up, with three parallel experiments per group. The experimental results are shown in Table 3. The optimal conditions were examined using Design Expert software, and a predictive regression model was established using the following quadratic regression equation: y = 17.89 + 2.17A - 1.57B + 1.3C + 0.51D + 0.96AB - 0.79AC - 0.45A - 0.38BC - 0.38BD + 0.12CD - 2.32A 2 -1.71B 2 -0.48C 2 -1.51D.

[0127] Table 3 Box-Behnken test design and results

[0128]

[0129]

[0130] The results of variance, goodness of fit, and adequacy analysis of the response surface model are shown in Table 4. The p-value (<0.0001) and F-value extraction time (159.2) confirmed the significance of the regression model, and the p-value (1.0) and F-value (0.0164) for lack of fit indicated that the prediction model was sufficient to accurately represent the test data. The coefficient of determination (R 2 ) is 0.9938, indicating that 99.38% of the variables can be determined by the model. 2 Adj =0.9918), indicating that the model can predict most of the variation in extraction rate. The smaller the P value, the more significant the corresponding coefficient. Based on the P value level, the linear coefficient and the quadratic coefficient (except CD) were all significant (p < 0.05).

[0131] Table 4 Analysis of variance of regression model

[0132]

[0133]

[0134] Note: “*” indicates significant (p<0.05); “**” indicates extremely significant (p<0.01); “***” indicates (p<0.001) and “ns” indicates not significant (p>0.05).

[0135] 2.3 Response surface analysis

[0136] The three-dimensional response surface plot and the two-dimensional response contour plot provide a visual explanation of the interaction between independent variables. The two-dimensional response contour plot can reflect the interaction between independent factors and show the significance of each independent factor. The circular response contour line indicates that the interaction between the corresponding variables is negligible, while the elliptical response contour line indicates that the interaction between the corresponding variables is significant. Figure 6 and Figure 7 As can be seen, the three-dimensional curves for AB, AC, AD, BC, and BD exhibit a steep trend, while the two-dimensional profiles are elliptical, indicating that these two interacting factors significantly influence the extraction yield of Camellia oleifera polysaccharides. The interaction between the liquid-to-solid ratio and water content is not significant. Extraction time has the greatest impact on the extraction efficiency. This effect can be explained by the fact that sufficient extraction time facilitates the separation of biopolymers from plant cells, while excessive extraction time may lead to dissolution of the product and other impurities, thereby reducing the extraction yield. Overall, the four parameters tested play a significant role in the extraction of Camellia oleifera polysaccharides. Further analysis determined the optimal extraction conditions for Camellia oleifera polysaccharides: a liquid-to-solid ratio of 14.38 mL / g, an extraction temperature of 71.32°C, an extraction time of 68.98 min, a water content of 29.63%, and a Camellia oleifera polysaccharide extraction yield of 19.10%. Based on actual results, the predicted conditions were slightly modified to: a liquid-to-solid ratio of 14.4 mL / g, an extraction temperature of 71°C, an extraction time of 69 min, and a water content of 30%. Under these conditions, three parallel experiments were conducted to validate the model, and the average extraction yield was 19.01±0.01%, slightly lower than the predicted value, indicating that the regression model is accurate and can be used to predict the extraction of polysaccharides from Camellia oleifera. Finally, we obtained a model suitable for optimizing the extraction process of DESs.

[0137] Example 3 Analysis of physicochemical properties of CLPs

[0138] 1. Experimental methods

[0139] 1.1 Determination of total sugar content

[0140] The neutral sugar content of CLP-W and CLP-D was determined using the phenol-sulfuric acid method. Different volumes (0, 0.2, 0.4, 0.6, 0.8, and 1 mL) of a 0.1 mg / mL glucose standard solution were pipetted into 10 mL EP tubes and made up to 1 mL with deionized water. 1 mL of 6% phenol solution was added to each tube and shaken thoroughly. Finally, 5 mL of concentrated sulfuric acid was added. After shaking thoroughly, the tubes were placed in a boiling water bath for 10 minutes, cooled to room temperature, and the OD value was measured at 490 nm. A standard curve was plotted with the corresponding OD value as the ordinate and the standard glucose content as the abscissa.

[0141] Weigh 2 mg of each CLP-W and CLP-D into a 10 mL volumetric flask and dilute to volume with deionized water. Then, pipette 1 mL of the polysaccharide solution and measure the absorbance of the sample at 490 nm according to the above procedure. Substituting the absorbance into the regression equation of the standard curve, the total sugar content in CLP-W and CLP-D was calculated using the following formula: Total sugar content (%) = (C × V × X / m) × 100%. Where V represents the volume of the extraction solvent (mL), m represents the sample weight (g), C represents the total sugar concentration in the sample, and X represents the dilution factor.

[0142] 1.2 Protein content determination

[0143] The protein content in CLP-W and CLP-D was determined using the Coomassie Brilliant Blue method. A standard protein solution with a concentration of 100 μg / mL was accurately prepared and stored at 4°C (using bovine serum albumin as the standard). 0.1-0.5 mL of the protein standard solution was placed in a test tube. Water was added to each tube to make it 1 mL, followed by the addition of 5 mL of Coomassie Brilliant Blue G-250 and thorough shaking. Distilled water was used as a blank control in place of the sample solution. After 15 minutes of immersion at room temperature, the absorbance was measured at 595 nm. A standard curve was constructed using the corresponding OD value as the vertical axis and the standard protein content as the horizontal axis.

[0144] Weigh approximately 5 mg of CLP-W and CLP-D into a 10 mL volumetric flask and dilute to volume with deionized water. Draw up 1 mL of the polysaccharide solution and measure the absorbance of the sample at 595 nm according to the above procedure. Substituting the absorbance into the regression equation of the standard curve, calculate the protein content of CLP-W and CLP-D. Protein content is calculated using the following formula: Protein content (%) = (C × V × X / m) × 100%. Where V represents the volume of the extraction solvent (mL), m represents the sample weight (g), C represents the protein concentration in the sample, and X represents the dilution factor.

[0145] 1.3 Determination of uronic acid content

[0146] Uronic acid content in CLP-W and CLP-D was determined using the m-hydroxybiphenyl colorimetric method. Prepare a 1 mg / mL galacturonic acid standard solution. Dissolve 0.478 g of sodium tetraborate in 100 mL of concentrated sulfuric acid to prepare a sulfuric acid-sodium tetraborate solution. Weigh 0.15 g of m-hydroxybiphenyl and dissolve it in 5 mg / mL sodium hydroxide solution, then dilute to 100 mL. Dilute the 1 mg / mL galacturonic acid standard to 5% to 35% concentrations. Transfer 100 μL of each solution to a test tube and add 1 mL of sulfuric acid-sodium tetraborate. Incubate at 95°C for 10 minutes. After cooling, add 10 μL of the m-hydroxybiphenyl reagent. Shake well and allow to develop color for 30 minutes. Measure the OD value at a wavelength of 525 nm. Replace the sample with distilled water as a blank control. Plot the curve with the OD value on the y-axis and the uronic acid content on the x-axis.

[0147] Weigh approximately 10 mg of each of CLP-W and CLP-D into a 10 mL volumetric flask and bring to volume with deionized water. Draw up 100 μL of the polysaccharide solution and measure the absorbance of the sample at 525 nm according to the above procedure. Substitute the absorbance into the regression equation of the standard curve to calculate the uronic acid content in CLP-W and CLP-D. The uronic acid content is calculated using the following formula: uronic acid content (%) = (C × V × X / m) × 100%. Where V represents the volume of the extraction solvent (mL), m represents the sample weight (g), C represents the uronic acid concentration in the sample, and X represents the dilution factor.

[0148] 1.4 Determination of total polyphenols content

[0149] The total phenol content was determined using the Folin-phenol method. The specific procedure was as follows: 500 μL of deionized water and 500 μL of a 0.5 mg / mL solution of Camellia oleifera polysaccharide were mixed in a 2 mL EP tube. 100 μL of a 10% Folin-phenol diluent was added and mixed thoroughly, followed by 300 μL of a 10% anhydrous sodium carbonate solution. The mixture was incubated in the dark for 1 hour. The absorbance of the reaction solution was measured at 750 nm. A curve was plotted using a gallic acid solution prepared in 80% methanol, and the sample absorbance was substituted into a standard curve using gallic acid as the standard to calculate the total phenol content. The total polyphenol content was calculated using the following formula: Total polyphenol content (%) = (C × V × X / m) × 100%. Where V represents the volume of the extraction solvent (mL), m represents the sample weight (g), C represents the polyphenol concentration in the sample, and X represents the dilution factor.

[0150] 1.5 Molecular weight determination

[0151] The molecular weight of camellia oleifera polysaccharides was determined by high performance gel permeation chromatography (HPGPC).

[0152] Sample preparation: Weigh 5 mg of standard and sample and dissolve them in DMSO to make a sample solution with a concentration of 1 mg / mL. Filter through a 0.22 μm filter membrane and then detect on the instrument.

[0153] Chromatographic conditions: The chromatographic column was a series connection of SHODEX KS-802 and KS-804 (7.8 mm × 300 mm); the column temperature was maintained at 40°C; the injection volume was 20 μL; the mobile phase was 0.2 mol / mL NaCl solution, and the flow rate was 0.8 mL / min.

[0154] 1.6 Determination of monosaccharide composition

[0155] High performance gas chromatography (GC-MS) was used to analyze the monosaccharide composition of camellia oleifera polysaccharides.

[0156] Sample preparation: 5 mg of sample and 1 mL of 2 mol / L trifluoroacetic acid (TFA) were placed in a pear-shaped flask, sealed with nitrogen, and hydrolyzed at 121°C for 2 h. 150 μL of 10 monosaccharide standards at 5 mM were mixed with the hydrolyzed polysaccharide and 150 μL of 1 mol / L NaOH solution.

[0157] HPLC conditions: A liquid column (Dionex CarboPac PA20, 150 × 3.0 mm, 10 μm) was maintained at 30°C. Mobile phases A (H₂O), B (0.1 mol / L NaOH), and C (0.1 mol / L NaOH, 0.2 mol / L NaCH₃COO) were used at a flow rate of 0.5 mL / min. Monosaccharide composition and quantification were determined by comparing peak area integration values and retention times of CLP-W and CLP-D with a calibrated standard curve using Chromeleon.

[0158] 1.7 Infrared spectroscopy

[0159] 2 mg of dried CLP-W and CLP-D samples were mixed with KBr in an agate mortar, ground, pressed into transparent sheets, and analyzed using a Fourier transform infrared spectrometer at 400-4000 cm -1 scanning.

[0160] 1.8 Differential Calorimetry

[0161] 2 mg of each CLP-W and CLP-D were weighed and sealed in an Al pan for DSC analysis. Measurements were performed under a nitrogen atmosphere at a rate of 10°C / min over a temperature range of 20–300°C. An empty pan was used as a control, and indium metal was used for calibration.

[0162] 1.9 Thermogravimetric analysis

[0163] Thermogravimetric analysis was performed using a TA instrument (TG209F3, Metter Toledo Corp., Zurich, Switzerland). 3 mg of CLP-W and CLP-D were placed in a dry ceramic crucible and weighed under nitrogen. The temperature range was 30–600°C at a heating rate of 20°C / min.

[0164] 1.10 Scanning electron microscopy observation

[0165] The polysaccharide was glued to the sample holder, and then the floating sample was blown away with an ear bulb, and then gold was sprayed and observed by scanning electron microscopy.

[0166] 1.11 Determination of emulsification properties

[0167] The emulsification capacity and emulsification stability of camellia oil leaf polysaccharides were determined by turbidity method. The specific operation is as follows: 10 mg / mL CLP-W and CLP-D solutions were prepared with distilled water and mixed with camellia oil at a volume ratio of 1:1. The water-oil mixture was used as a control. The solution was homogenized at 10,000 rpm for 1 min. Then, 0.2 mL of emulsion was immediately pipetted from the bottom of the container, and after 10 minutes, it was mixed with 0.8 mL of sodium dodecyl sulfate (0.1% w / v) solution. Subsequently, the absorbance of the diluted emulsion was measured at 500 nm using a spectrophotometer (SpectraMax M2, Molecular Devices, USA). The formula for calculating the emulsification activity index and emulsion stability is as follows: Emulsification activity index Where A0 is the absorbance recorded immediately, the dilution factor (DF) is 5, C is the CLPs concentration (g / mL), is the oil volume fraction (0.5). Emulsion stability = (A0 × ΔT) / (A0 - A10). Where A0 is the absorbance recorded immediately, A10 is the absorbance recorded 10 minutes after emulsification, and ΔT is the time interval.

[0168] In addition, the droplet size of the prepared emulsions was measured using a confocal laser scanning microscope (FV1200, Olympus Corporation, Japan). The initial emulsion was diluted with distilled water and Nile Red fluorescent dye was added before measurement. The zeta potential and particle size of the emulsions were then investigated using a dynamic optical nanoparticle potentiostat (Zetasizer Nano-ZS, Malvern Instruments, UK).

[0169] 2. Experimental results

[0170] 2.1 Chemical composition analysis

[0171] The chemical composition (total sugar, protein, uronic acid, and total polyphenol content) of hot-water-extracted Camellia oleifera polysaccharides (CLP-W) and DES-3-extracted Camellia oleifera polysaccharides (CLP-D) was determined, and the results are shown in Table 5. The total sugar content of CLP-D and CLP-W was 86.58 ± 1.04% and 80.83 ± 1.01%, respectively. Protein content was 3.59 ± 0.13% for CLP-D and 1.58 ± 0.12% for CLP-W, and 5.91 ± 0.56% for CLP-D and 4.68 ± 0.28% for CLP-W. Furthermore, the uronic acid content of CLP-D was slightly higher at 18.32 ± 0.12% than that of CLP-W (17.60 ± 1.90%).

[0172] Table 5 Physicochemical properties and monosaccharide composition of CLP-W and CLP-D

[0173]

[0174] Note: Fuc, trehalose; Man, mannose; Rha, rhamnose; Gal-A, galacturonic acid; Glc, glucose; Gal, galactose; Xyl, xylose; Ara, arabinose; Glc-A, glucuronic acid.

[0175] 2.2 Molecular weight analysis

[0176] The molecular weights of CLP-D and CLP-W were determined by HPGPC. The results are shown in Table 4. The molecular weight (Mw) of CLP-D was 96.56 KDa, which was significantly smaller than that of CLP-W (775.76 KDa).

[0177] 2.3 Monosaccharide composition analysis

[0178] The monosaccharide composition of CLP-D and CLP-W was determined by GC-MS by comparing the retention time and peak area with those of the standard. The results are shown in Table 4 and Figure 8 As shown in Table 4, CLP-D and CLP-W have the same monosaccharide composition types but different molar ratios. Nine monosaccharides, including trehalose, arabinose, rhamnose, glucose, galactose, xylose, mannose, galacturonic acid, and glucuronic acid, were detected in both CLP-D and CLP-W. Among them, the main monosaccharide compositions of the two were arabinose, rhamnose, glucose, and galactose, with a proportion close to 90%. In addition, the zeta potential, particle size, conductivity, and viscosity results of CLP-D and CLP-W are also shown in Table 4. The slight differences between them can be attributed to different chemical compositions and monosaccharide compositions. In particular, the molecular weight and particle size of CLP-D are smaller than those of CLP-W, which may be because the hydrogen bonds in DESs perform better in breaking the molecular bonds of biopolymers.

[0179] 2.4 Infrared spectroscopy analysis

[0180] Infrared spectroscopy is widely used for the qualitative determination of organic functional groups in polysaccharides. In this study, the infrared spectra of CLP-D and CLP-W both showed characteristic peaks of polysaccharides, but with slight differences. Figure 8 As shown in B. At 3432.4cm -1 The vibration band at 2930cm is due to the OH stretching vibration of polysaccharide. -1 The peak near 1635.1cm is the CH stretching vibration. -1 The strong absorption band at 1731.18 cm is defined as the characteristic of asymmetric stretching vibration of carboxyl (-COOH) and aldehyde (-CHO) groups of aldehyde acid. -1 There is another obvious absorption peak at 1453.6 cm, which can be interpreted as the characteristic of the stretching vibration of the ester carbonyl (C=O), indicating that CLP-D contains O-acetyl groups. The stretching vibration of the CH bond produces a peak at 1453.6 cm -1 (CLP-D) and 1418.9cm -1 (CLP-W) absorption peak. In addition, 1040.4 cm -1 (CLP-D) and 1067.6cm -1 The absorption peak at (CLP-W) is defined as the COC stretching vibration on the pyranose ring, indicating that the two contain abundant glycosidic bonds. -1 (CLP-D) and 709.2cm -1 The characteristic absorption of D-pyranose was observed at the absorption peak of (CLP-W). -1 (CLP-D) and 613.7cm -1 The peak at (CLP-W) is the C-C stretching vibration. In summary, both CLP-D and CLP-W are pyranose.

[0181] 2.5 Thermal stability analysis

[0182] The thermal stability of polysaccharides is a key factor that should be considered in their application. The DSC curve can reflect the changes in the thermal properties of polysaccharides as the temperature continues to rise. Figure 8 As shown in Figure C, due to the loss of free water and bound water, the DSC curves of CLP-D and CLP-W both show a broad endothermic peak in the range of 100-150°C, and the peak temperature of CLP-D is higher than that of CLP-W. However, neither of them has an endothermic peak in the range of 200-300°C. It can be inferred that the glass transition temperature (Tg) of the polysaccharide may be after 300°C. The thermal decomposition law and thermal stability of tea oil tea polysaccharides were determined by thermogravimetric analyzer. The mass loss of the sample is shown in Figure 2. Figure 8As shown in Figure 3D, the TGA curves clearly show a decrease in weight for both CLP-D and CLP-W from room temperature to 200°C, which can be explained by the departure of bound water from the polysaccharide structure. Importantly, at the same temperature, the weight loss rate of CLP-W is higher than that of CLP-D, indicating that CLP-D possesses better thermal stability than CLP-W. In summary, both CLP-W and CLP-D polysaccharides exhibit excellent thermal properties, but CLP-D performs better, which provides greater opportunities for its widespread application in food processing and production.

[0183] 2.6 Scanning electron microscopy analysis

[0184] The cell fragmentation of plants is positively correlated with the extraction rate of active substances. The more severe the fragmentation, the higher the extraction rate. The effects of different extraction methods on extraction efficiency were further evaluated by scanning electron microscopy of the tea leaf powder residue after extraction with different solvents. The SEM images of tea leaf powder, residue after hot water and DES-3 extraction, CLP-D and CLP-W are shown in Figure 2. Figure 9 As shown in the figure, it is clearly seen that the cell wall damage of samples treated with DES-3 is greater than that of samples treated with hot water, providing strong evidence for the high extraction efficiency of DESs as green solvents. The apparent structures of CLP-D and CLP-W are irregular flakes and block structures, respectively, which may be one of the reasons for their different functions and bioactivities.

[0185] 2.7 Emulsification ability analysis

[0186] Polysaccharides exhibit good emulsification ability due to their exposed hydrophobic groups. Figure 10 As shown. Among them, Figure 10 Figures AB show fresh emulsions prepared at room temperature at a concentration of 10 mg / mL for CLP-W and CLP-D, and emulsions stored for 30 days. The results show that the two emulsions clearly separated during storage, and the bottom layer of the CLP-D emulsion was smaller than that of the CLP-W emulsion, indicating that CLP-D has better emulsification ability. In addition, we also measured the droplet size, emulsification activity and emulsification stability, zeta potential, and particle size of the emulsions. Figure 10 CG. The results showed that compared with CLP-W, CLP-D emulsions had smaller droplet and particle sizes, but higher absolute zeta potentials. This is likely due to CLP-D's lower molecular weight and higher protein content. The emulsion activity index (EAI) and emulsion stability index (ESI) indicate the ability of polysaccharides to form stable emulsions. Fresh emulsions prepared with CLP-D exhibited significantly higher EAI and ESI values than those prepared with CLP-W (p < 0.001). In summary, CLP-D is more suitable as an emulsifier in food production.

[0187] Example 4 Protective Effects of CLPs on Caenorhabditis elegans Induced by Heavy Metals

[0188] 1. Experimental methods

[0189] 1.1 LC of different heavy metals 50 analyze

[0190] Using the time-limited egg-laying method, nematodes were cultured synchronously on blank NGM culture plates to the L4 stage. Twenty Caenorhabditis elegans were exposed to a 24-well plate containing liquids of different concentrations of heavy metals (Al, Hg, Pb, Cd, Cr, Ni) for 24 hours to induce acute poisoning. The head or tail of Caenorhabditis elegans was gently touched with a platinum wire. If there was no response, it was recorded as death, and the number of deaths, survivors and totals were counted. At least three independent tests were performed. The half-lethal concentration (LC) was calculated using IBM SPSS 20 statistical software. 50 .

[0191] 1.2 Determination of CLPs Dosage Concentration

[0192] Synchronized L4-stage nematodes were transferred to 24-well plates containing Hg and tea oil tea polysaccharides. After 24 hours of treatment, the number of surviving C. elegans and the total number of C. elegans were recorded, and the survival rate was calculated. The optimal polysaccharide treatment concentration was ultimately screened. Wells containing K solution alone served as blank controls. Three independent experiments were performed with at least 20 C. elegans in each group.

[0193] 1.3 Effects of CLPs on the survival rate of Caenorhabditis elegans under heavy metal stress

[0194] Use the screened LC (Al, Hg, Pb, Cd, Cr, Ni) 50 The number of surviving and total nematodes in the L4 stage of Caenorhabditis elegans treated with the optimal tea oil polysaccharide was recorded 24 hours later. The survival rate was calculated, with wells containing only K solution as blank controls. Each group had at least 20 nematodes, and each experiment was repeated three times.

[0195] 2. Experimental results

[0196] 2.1 Heavy Metal LC 50 analyze

[0197] Different concentrations of Hg, Al, Pb, Cd, Cr, and Ni solutions were prepared and subjected to a 24-hour acute exposure test on L4 stage Caenorhabditis elegans. The total number, number of deaths, and number of survivors were counted, and the half-lethal concentration (LC50) of each heavy metal was calculated using IBM SPSS Statistics 20. 50 ), LC 50 The results are shown in Table 6. 50 The minimum is 83.11 μM, which means that Hg is the most toxic among the heavy metals used in this experiment.

[0198] Table 6 LC values of different heavy metals 50 value

[0199]

[0200] 2.2 Analysis of the optimal concentration of CLPs

[0201] Select the LC of the most toxic heavy metal Hg 50 The concentration screening test of CLP-W and CLP-D was carried out. CLP-W and CLP-D solutions with a concentration of 2 mg / L were prepared with K solution and mixed with an equal volume of 2 times LC 50 The Hg solution was mixed to prepare the final concentrations of CLP-W and CLP-D at 0, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 mg / mL. The L4 stage Caenorhabditis elegans was subjected to a 24-hour acute exposure test. The number of surviving and total nematodes was recorded and the survival rate was calculated. Figure 11 As shown in Figures AB, the highest survival rates of C. elegans under Hg stress were 78.7 ± 2.34% and 81.1 ± 2.17% at 0.3 mg / mL CLP-W and CLP-D, respectively. Therefore, 0.3 mg / mL CLP-W and CLP-D were selected for subsequent experiments.

[0202] 2.3 CLPs improve the survival rate of Caenorhabditis elegans under heavy metal stress

[0203] The heavy metal LC 50 The L4 stage Caenorhabditis elegans was exposed to the best concentrations of CLP-W and CLP-D for 24 hours to evaluate the mitigating effect of Camellia oleifera polysaccharides on the damage of Caenorhabditis elegans caused by different heavy metals. The results are as follows Figure 12 As shown. Both CLP-W and CLP-D significantly improved the survival rate of C. elegans under stress of heavy metals Al, Cr, Cd, Pb, Ni, and Hg (p < 0.0001). Among them, the best mitigation effect was on Al, with CLP-W and CLP-D significantly increasing the survival rate of nematodes under Al stress by 25.16% and 25.51%, respectively (p < 0.001). The addition of CLP-W and CLP-D increased the survival rate of C. elegans by 15.19% and 23.37%, respectively, compared with the Cr treatment group, 18.97% and 19.53% compared with the Cd treatment group, 22.64% and 22.99% compared with the Pb treatment group, 16.47% and 14.72% compared with the Ni treatment group, and 17.77% and 23.49% compared with the Hg treatment group. In general, CLP-D is better than CLP-W in improving the survival rate of C. elegans under heavy metal stress.

[0204] Furthermore, this example demonstrates, through experiments with Caenorhabditis elegans, that CLP-D extracted using the method provided by the present invention enhances the nematode's resistance to heavy metal stress by regulating the nuclear translocation of DAF-16, SKN-1, and HSF-1 nuclear transcription factors, thereby activating the nematode's oxidative defense system, reducing ROS and MDA levels in the nematode, reducing lipid and lipofuscin accumulation, and alleviating intestinal damage. These results confirm that Camellia oleifera polysaccharides have the ability to mitigate the toxicity of various heavy metals.

Claims

1. A method for extracting oil tea polysaccharides based on a deep eutectic solvent, characterized in that: The method comprises the following steps: (1) drying and crushing oil tea leaves to obtain oil tea leaf powder; (2) extracting the camellia oleifera powder described in step (1) using a deep eutectic solvent Bet-LA to obtain a camellia oleifera polysaccharide extract; (3) subjecting the oil tea leaf polysaccharide extract described in step (2) to alcohol precipitation, collecting the precipitate, and freeze-drying to obtain oil tea leaf polysaccharide; In the deep eutectic solvent Bet-LA, the hydrogen bond acceptor is betaine and the hydrogen bond donor is lactic acid; The molar ratio of betaine to lactic acid in the deep eutectic solvent Bet-LA is 1:(1-5); The water content of the deep eutectic solvent Bet-LA is 10%-50%; The liquid-to-solid ratio of the deep eutectic solvent Bet-LA to the oil tea leaf powder in step (2) is (5-25) mL / g; The extraction temperature in step (2) is (60-80)°C; The extraction time in step (2) is (60-80) min.

2. The method according to claim 1, characterized in that The molar ratio of betaine to lactic acid in the deep eutectic solvent Bet-LA is 1:

3.

3. The method according to claim 1, characterized in that The water content of the deep eutectic solvent Bet-LA is 30%.

4. The method according to claim 1, wherein The liquid-to-solid ratio of the low eutectic solvent Bet-LA and the oil tea leaf powder in step (2) is 14.4 mL / g.

5. The method according to claim 1, wherein The extraction temperature in step (2) is 71°C.

6. The method according to claim 1, wherein The extraction time in step (2) was 69 min.

7. The method according to claim 1, characterized in that The extraction method in step (2) is constant temperature water bath extraction.

8. The method according to claim 1, characterized in that The alcohol precipitation in step (3) is carried out using ethanol.

9. The method according to claim 1, characterized in that After collecting the precipitate in step (3), the precipitate is further treated as follows: Sevag reagent is added to the precipitate to remove protein, and the supernatant is collected.

10. The method according to claim 9, characterized in that The Sevag reagent is a reagent containing n-butanol and chloroform.

11. The method according to claim 10, characterized in that The ratio of n-butanol to chloroform in the Sevag reagent is 1:

4.

12. The method according to claim 9, characterized in that The freeze-drying step in step (3) further includes the following steps: dialyzing the supernatant to remove the low eutectic solvent Bet-LA.

13. Camellia oleifera polysaccharide prepared by the method according to any one of claims 1 to 12.

14. Use of the oil tea leaf polysaccharide according to claim 13 in the preparation of a medicament for alleviating heavy metal toxicity or treating heavy metal poisoning.

15. The use according to claim 14, characterized in that The heavy metals include Hg, Pb, Cd, Cr and / or Ni.

16. Use of the method according to any one of claims 1 to 12 in extracting polysaccharides from camellia oleifera.

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