Method for extracting oil tea fruit shell having lipase inhibitory activity xylan by using deep eutectic solvent

Abstract

The application belongs to the technical field of natural product extraction and functional polysaccharide preparation, and discloses a method for extracting oil tea fruit shell xylan with lipase inhibitory activity by using a deep eutectic solvent, which comprises the following steps: (1) raw material pretreatment; (2) deep eutectic solvent preparation; (3) extraction reaction; and (4) precipitation and purification. The application provides a method for selectively extracting oil tea fruit shell xylan with lipase inhibitory activity by using an alkaline deep eutectic solvent, the method has mild extraction conditions, the structure of the extracted xylan is complete, the product has significant pancreatic lipase inhibitory activity, the solvent can be recycled and used, the method is green and environment-friendly, and a new way is provided for high-value utilization of oil tea fruit shell by-products.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and functional polysaccharide preparation technology, specifically involving a green preparation method for selectively extracting xylan with lipase-inhibiting activity from Camellia oleifera fruit shell using a deep eutectic solvent (DES). Background Technology

[0002] Camellia oleifera Abel is a unique woody oil crop in my country, with a wide planting area, producing a large amount of camellia fruit shells (tea husks) as a byproduct every year. The hemicellulose content in camellia fruit shells is as high as 36%~50%, far exceeding that of ordinary wood and gramineous raw materials. Xylan is the main component of hemicellulose and has great potential for high-value utilization. Currently, most camellia fruit shells are burned or discarded, causing both resource waste and environmental pressure. Extraction of polysaccharides from camellia often focuses on water-soluble polysaccharides, with limited research on xylan, the main component of camellia (Yu Yanling. 2025). (Yu Yanling. Research on the fractionation and high-value utilization of components in camellia fruit shells [D]. Anhui University of Technology, 2025. DOI:10.27763 / d.cnki.gahgc.2025.000636). Research on xylan from Camellia oleifera mainly focuses on the preparation of oligoxylans, while there is a lack of research on the extraction, separation, and activity identification of high-polyxylans with complete structure and activity (Chen Zhanyan. 2026) (Chen Zhanyan, Xu Sijia, Huang Yixin, et al. Research on key processes for the preparation of xylan oligosaccharides from Camellia oleifera fruit shells by steam flash explosion [J / OL]. China Oils and Fats, 1-17 [2026-07-12]).

[0003] Xylan is a heteropolysaccharide whose main chain is composed of β-D-xylanose linked by β-(1→4) glycosidic bonds, with side chains often linked to L-arabinose, 4-O-methyl-D-glucuronic acid, and other groups. Studies have shown that natural polysaccharides have good lipase inhibitory activity, and their mechanism of action is closely related to their high molecular weight and high viscosity: high-viscosity polysaccharides can form a physical barrier in the gastrointestinal tract, hindering the contact between lipase and lipid substrates; at the same time, polysaccharides can disrupt the stable lipid emulsion interface of bile salts, inhibiting lipase function, thereby reducing the digestion and absorption of dietary fat, and have good application prospects in the fields of weight loss and lipid-lowering functional foods.

[0004] Traditional methods for xylan extraction mainly include alkaline extraction, hot water extraction, and DMSO extraction (Tang Ning. 2023) (Tang Ning. Study on the Composition and Structure of Hemicellulose from Camellia oleifera Fruit Shell [D]. South China University of Technology, 2023. DOI:10.27151 / d.cnki.ghnlu.2023.004344). Alkaline extraction causes serious environmental pollution and easily leads to polysaccharide degradation and structural damage; hot water extraction has low efficiency and long processing time; DMSO extraction is costly and highly toxic. In existing technologies, eutectic solvents have been used as novel green solvents for the separation of lignocellulose components, but there are no reports on the selective extraction of xylan from Camellia oleifera fruit shell and its targeted preparation for inhibiting lipase activity.

[0005] Current research on DES extraction of hemicellulose from Camellia oleifera fruit shells mainly focuses on the crystallinity, rheological properties, and thermal stability of the product, without addressing the targeted regulation of biological activities such as lipase inhibition. Furthermore, conventional acidic DES easily causes polysaccharide backbone breakage, leading to a decrease in molecular weight, which is detrimental to the preservation and exertion of its lipase inhibitory activity. Therefore, developing a green and efficient extraction method that can completely preserve the higher-order structure and biological activity of xylan is of great significance for the high-value utilization of Camellia oleifera fruit shell resources. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for selectively extracting xylan with lipase-inhibiting activity from Camellia oleifera fruit shells using an alkaline eutectic solvent. This method employs mild extraction conditions, preserves the complete xylan structure, and produces a product with significant pancreatic lipase-inhibiting activity. Furthermore, the solvent can be recycled, making it environmentally friendly and providing a new avenue for the high-value utilization of Camellia oleifera fruit shell byproducts.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for extracting xylan with lipase-inhibiting activity from Camellia oleifera fruit shell using a eutectic solvent includes the following steps: (1) Raw material pretreatment: The camellia fruit shells are washed, dried, crushed, and passed through a 60-80 mesh sieve to obtain camellia fruit shell powder; the wax and fat-soluble impurities are removed by Soxhlet extraction with toluene-ethanol mixture, and then dried for later use.

[0008] (2) Preparation of eutectic solvent: Choline chloride is used as hydrogen bond acceptor and monoethanolamine is used as hydrogen bond donor. They are mixed in a molar ratio of 1:2 to 1:8 and heated and stirred at 60 to 90°C until a uniform and transparent liquid is formed, which yields choline chloride / monoethanolamine eutectic solvent (ChCl / MEA DES).

[0009] (3) Extraction of xylan by DES: The pretreated camellia fruit shell powder is mixed with the above eutectic solvent at a solid-liquid ratio of 1:10~1:30 (g / mL), and mechanically stirred at 60~100℃ for 30~60 min. After extraction, the mixture is cooled to room temperature, and 2~4 times the volume of deionized water is added to dilute it. The supernatant is then collected by centrifugation.

[0010] (4) Xylan precipitation recovery: Add anhydrous ethanol to the supernatant until the final volume fraction of ethanol is 60%~80%, let it stand at 4℃ for 12~24h to precipitate; collect the precipitate by centrifugation, wash repeatedly with anhydrous ethanol, dialyze through a dialysis bag to remove salt, freeze dry to obtain the xylan product from Camellia oleifera fruit shell.

[0011] (5) DES solvent recovery: Collect the supernatant of ethanol after precipitation, recover ethanol and water by vacuum distillation, concentrate the remaining aqueous phase and add an appropriate amount of monoethanolamine and water, and the regenerated eutectic solvent can be reused for extraction.

[0012] Furthermore, the preferred molar ratio of choline chloride to monoethanolamine in step (2) is 1:3, and the preferred heating temperature is 80°C.

[0013] Furthermore, the solid-liquid ratio in step (3) is preferably 1:20 (g / mL), the extraction temperature is preferably 80℃, and the extraction time is preferably 45min.

[0014] Furthermore, mechanical stirring extraction can be used in step (3) at a rate of 500 r / min.

[0015] Furthermore, the deionized water dilution factor in step (3) is 2 to 4 times the volume of DES.

[0016] Furthermore, the dialysis in step (4) uses a dialysis bag with a molecular weight cutoff of 3500 Da.

[0017] Furthermore, the final volume fraction of ethanol in step (4) is preferably 70%.

[0018] Furthermore, the regenerated DES described in step (5) can be reused in the extraction process of step (3) for no less than 3 cycles.

[0019] Furthermore, the prepared Camellia oleifera fruit shell xylan has a purity of ≥75%; and its inhibition rate against pancreatic lipase is ≥65% at a concentration of 2 mg / mL.

[0020] The beneficial effects of this invention are: 1. It has good structural integrity and excellent lipase inhibitory activity. This invention, by strictly controlling the molar ratio of ChCl to MEA (1:2~1:4) and the extraction temperature, effectively avoids the β-(1→4)-glycosidic bond breakage caused by the strong alkaline environment in traditional alkaline extraction methods while ensuring hemicellulose dissolution. This polysaccharide possesses both physical barrier effects (increasing system viscosity to hinder substrate diffusion) and interfacial disruption effects (side chain groups interfering with enzyme-substrate binding). At a concentration of 2 mg / mL, it achieves an inhibition rate of 45%~65% against pancreatic lipase, significantly higher than products obtained through conventional hot water extraction, and can be used as a highly effective natural lipid-lowering factor.

[0021] 2. High extraction specificity and high product purity. The ChCl / MEA DES system exhibits a specific cleavage effect on the ester bonds in the lignin-carbohydrate complex (LCC), efficiently dissociating the bond between hemicellulose (xylan) and lignin, while having a weak degradation effect on cellulose. This characteristic results in extremely low lignin residue in the extract; after a single ethanol precipitation and dialysis purification, the xylan purity can reach over 75%, simplifying subsequent purification processes.

[0022] 3. The process is green and environmentally friendly, and the solvent can be recycled. This invention eliminates the use of volatile and toxic organic solvents (such as benzene and pyridine) in traditional processes, instead employing a biocompatible eutectic solvent. More importantly, addressing the challenges of DES recovery, this invention achieves efficient solvent regeneration through a strategy of "recovering ethanol by vacuum distillation to replenish lost components." Experiments show that the regenerated DES can be recycled more than three times without a significant decrease in xylan extraction rate or product purity, greatly reducing production costs and waste emissions, aligning with the development concept of green chemistry.

[0023] 4. High raw material utilization rate and significant economic added value. This invention uses camellia fruit shells, a solid waste product from camellia oil processing, as raw material, solving the environmental problems caused by the accumulation and decay of these shells. Through DES technology, these shells are transformed into high-value-added medicinal / edible xylan concentrates, not only extending the camellia oil industry chain but also providing a new technological path for the high-value utilization of agricultural and forestry waste, resulting in significant economic and social benefits. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the overall process of extracting xylan with lipase-inhibiting activity from Camellia oleifera fruit shells according to the present invention. Figure 2 Fourier transform infrared (FT-IR) spectrum of xylan from Camellia oleifera fruit shell prepared in Example 2; Figure 3 The graph shows the change in the inhibition rate of pancreatic lipase by xylan from Camellia oleifera fruit shell prepared in Example 2. Figure 4 A comparative graph showing the effect of the number of times the eutectic solvent is recycled on xylan extraction rate and lipase inhibitory activity. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the content described.

[0026] Example 1 Raw material pretreatment: Clean camellia fruit shells were collected, removing branches and moldy parts. The shells were rinsed with deionized water to remove surface dust and sand. They were then dried in a 60℃ forced-air drying oven to constant weight, pulverized, and passed through a 50-mesh sieve. The sieved material was placed in a Soxhlet extractor, and a toluene-ethanol (2:1, v / v) mixture was added. The mixture was refluxed for 6 hours to remove wax and fat-soluble impurities. After extraction, the residue was removed, dried in a ventilated environment at 40℃ to evaporate residual solvent, and then sealed for storage to obtain pretreated camellia fruit shell powder.

[0027] Preparation of DES: Weigh choline chloride, the hydrogen bond acceptor, and monoethanolamine, the hydrogen bond donor, and place them in a round-bottom flask at a molar ratio of 1:3. Stir in a constant temperature water bath at 80°C (500 r / min) for about 30 min until a homogeneous, clear and transparent liquid is formed. This is the eutectic solvent of choline chloride / monoethanolamine (ChCl / MEA DES), which is then stored in a desiccator for later use.

[0028] Extraction process: Accurately weigh 5.0 g of pretreated Camellia oleifera fruit shell powder, add 100 mL of the above-mentioned DES (solid-liquid ratio 1:20, g / mL), and mix well. Place the mixture in an 80℃ constant temperature water bath and stir continuously (500 r / min) for 60 min. After the reaction is complete, immediately remove the mixture, add 300 mL of deionized water preheated to 60℃ to dilute and reduce the viscosity of the system, then centrifuge at 5000 r / min for 15 min to separate the precipitate and collect the supernatant containing xylan.

[0029] Precipitation and Drying: Slowly add pre-cooled anhydrous ethanol dropwise to the supernatant while stirring until the final ethanol volume fraction reaches 70%. Place the mixture in a 4°C refrigerator and allow it to precipitate for 16 hours. The next day, collect the precipitate by centrifugation at 8000 rpm for 20 minutes. Wash the precipitate three times with 70% ethanol solution to remove residual monoethanolamine and small molecule impurities. Redissolve the washed precipitate in an appropriate amount of deionized water and transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialyze the dialysis fluid for 48 hours. Finally, freeze-dry the dialysis fluid to obtain a white flocculent camellia oleifera fruit shell xylan-enriched product.

[0030] Results: The xylan extraction rate was 14.8% (based on the dry weight of the raw material), and the xylan purity was 78.3%; the inhibition rate against pancreatic lipase at a concentration of 2 mg / mL was 60.21%.

[0031] Example 2 The operation procedure in this embodiment is basically the same as that in Embodiment 1, except that: to explore the extraction effect under milder conditions, the proportion of hydrogen bond donors in the eutectic solvent and the extraction process parameters were adjusted in this embodiment. Details are as follows: The molar ratio of choline chloride to monoethanolamine was adjusted to 1:4, and the solid-liquid ratio was adjusted to 1:10 (g / mL). During the extraction process, the extraction temperature was set to 70℃, and the extraction time was extended to 120 min.

[0032] The xylan enrichment obtained in this example was tested and found to have the following properties: the xylan extraction rate was 11.2% based on the dry weight of the raw material; the xylan purity was 81.5%, which is an improvement over Example 1; and the inhibition rate of pancreatic lipase was 65.2% at a concentration of 2 mg / mL.

[0033] Compared to Example 1, this example, by lowering the extraction temperature, extending the extraction time, and appropriately increasing the proportion of monoethanolamine, although resulting in a slight decrease in the extraction rate, effectively reduced the random degradation of the xylan backbone by the strongly alkaline environment, thereby achieving higher product purity. Simultaneously, more complete side-chain structures (such as arabinose and glucuronic acid groups) were preserved, which may be the structural basis for its slightly higher lipase inhibition rate (51.2%) at the same concentration. This example demonstrates the feasibility of preparing highly active xylan under low-energy and mild conditions according to the present invention.

[0034] Example 3 The operation procedure in this embodiment is basically the same as that in Embodiment 1, except that: to verify the critical range of process parameters and the destructive effect of a strong alkaline and high-temperature environment on the product structure, this embodiment uses more extreme extraction conditions. Specifically: The molar ratio of choline chloride to monoethanolamine was adjusted to 1:8 (high alkaline system), and the solid-liquid ratio was adjusted to 1:30 (g / mL). During the extraction process, the extraction temperature was set to 100℃ and the extraction time was shortened to 30 min.

[0035] Results: Based on the dry weight of the raw material, the xylan extraction rate was 15.9%; the xylan purity was 72.4%; and the inhibition rate against pancreatic lipase at a concentration of 2 mg / mL was 49.6%.

[0036] Compared to Examples 1 and 2, although this example achieved the highest extraction rate by disrupting the cell wall structure using high temperature and high concentration of monoethanolamine, the excessively high reaction temperature (100°C) and extremely alkaline environment (molar ratio 1:8) triggered glycosidic bond breakage. Since the pancreatic lipase inhibitory activity is highly dependent on the molecular weight and spatial conformation of xylan, the structural disruption ultimately led to a significant decrease in its inhibition rate (39.6%).

[0037] Example 4: DES Recycling Performance The supernatant after ethanol precipitation in Example 1 was collected and distilled under reduced pressure at 45°C to recover ethanol and water. The remaining aqueous phase was concentrated to replenish the monoethanolamine and water lost through evaporation. This regenerated DES was then used repeatedly for the extraction of xylan from Camellia oleifera fruit shells under the same material-to-liquid ratio, temperature, and time conditions as in Example 1, for a total of 3 cycles.

[0038] With increasing cycles, the extraction efficiency of DES decreased slightly, but by the third cycle, the xylan extraction rate remained at 91.2% of the initial extraction rate. Furthermore, the total sugar purity and in vitro lipase inhibitory activity of the recycled products showed no significant difference compared to the first-extracted sample. This indicates that the DES structure is stable and possesses good recyclability and reuse potential.

[0039] Comparative Example 1: Traditional Alkali Extraction Take 5 g of pretreated Camellia oleifera fruit shell powder, add 100 mL of 10% NaOH solution at a material-to-liquid ratio of 1:20 (g / mL), and extract by stirring in an 80℃ constant temperature water bath for 3 h. After the reaction is complete, cool to room temperature, neutralize with concentrated hydrochloric acid to neutral, and then centrifuge at 5000 r / min for 15 min to collect the supernatant. The supernatant is precipitated with 3 volumes of anhydrous ethanol, dialyzed (molecular weight cutoff 3500 Da) to remove salt, and then freeze-dried to obtain the alkali-extracted xylan sample.

[0040] Under these conditions, the xylan extraction rate was 12.5%, and the product purity was 65.8%; at a concentration of 2 mg / mL, its inhibition rate against pancreatic lipase was 37.4%. Compared with Example 1 of this invention, the alkaline extraction rate and product purity were significantly reduced, and the biological activity was greatly diminished, indicating that the DES method described in this invention has significant advantages in terms of extraction efficiency and product activity retention.

[0041] Comparative Example 2: Acidic DES Extraction Extraction was performed using an acidic eutectic solvent (DES) consisting of choline chloride and malic acid (molar ratio 1:1), under the same material-liquid ratio, temperature, and time conditions as in Example 1.

[0042] The xylan extraction rate was 13.7%, with a purity of 68.2%. At a concentration of 2 mg / mL, its inhibition rate against pancreatic lipase was only 32.8%. The reason for this is that the H+ ions dissociated from malic acid catalyzed the acid hydrolysis of xylan glycosidic bonds under heating conditions, leading to severe degradation of the polysaccharide chains, a significant decrease in molecular weight, and consequently a substantial decline in the product's biological activity. This confirms the necessity of neutral DES in protecting the active conformation of xylan.

[0043] Methods for Assaying Lipase Inhibitory Activity The inhibitory activity of pancreatic lipase was determined using the p-nitrophenol palmitate (p-NPP) method: 0.1 mL of xylan solutions of different concentrations were taken, with orlistat as a positive control, and 0.1 mL of pancreatic lipase solution (1 mg / mL) was added and incubated at 37 °C for 10 min; 0.6 mL of Tris-HCl buffer (pH 7.4) and 0.2 mL of p-NPP substrate solution were added, and the reaction was carried out at 37 °C for 15 min; the absorbance value at 405 nm was measured, and the inhibition rate was calculated.

Claims

1. A method for extracting xylan with lipase-inhibiting activity from Camellia oleifera fruit shell using a eutectic solvent, characterized in that, Includes the following steps: (1) Raw material pretreatment: The camellia fruit shells are washed, dried, crushed and dewaxed to obtain camellia fruit shell powder; (2) Preparation of eutectic solvent: Choline chloride is used as hydrogen bond acceptor and monoethanolamine is used as hydrogen bond donor. They are mixed in a molar ratio of 1:2 to 1:8 and heated and stirred until a uniform transparent liquid is formed to obtain choline chloride / monoethanolamine eutectic solvent. (3) Extraction reaction: Mix the camellia fruit shell powder with a eutectic solvent at a solid-liquid ratio of 1:10~1:30 (g / mL), stir at 60~100℃ for 30~120 min, dilute with water after reaction, and centrifuge to take the supernatant. (4) Precipitation and purification: Add ethanol to the supernatant until the final volume fraction is 60%~80%, let it stand to precipitate, collect the precipitate, wash it, dialyze it through a dialysis bag with a molecular weight cutoff of 3500 Da, and dry it to obtain the xylan from the camellia fruit shell.

2. The method according to claim 1, characterized in that, In step (2), the molar ratio of choline chloride to monoethanolamine is 1:3 to 1:6, and the heating and stirring temperature is 60 to 90°C.

3. The method according to claim 1, characterized in that, In step (3), the solid-liquid ratio is 1:20 g / mL, the extraction temperature is 70~85℃, and the extraction time is 45-60min.

4. The method according to claim 1, characterized in that, In step (3), the amount of water used for dilution is 2 to 4 times the volume of the eutectic solvent.

5. The method according to claim 1, characterized in that, In step (4), the final volume fraction of ethanol is 70%, and the mixture is allowed to stand at 4°C for 12-24 hours to precipitate.

6. The method according to claim 1, characterized in that, It also includes step (5) solvent recovery and recycling: collect the supernatant after ethanol precipitation in step (4), recover ethanol and water by vacuum distillation, replenish the lost monoethanolamine and water, and obtain regenerated eutectic solvent. The regenerated eutectic solvent is returned to step (3) for recycling, and the number of cycles is not less than 3.

7. A type of xylan from Camellia oleifera fruit shell prepared by the method according to any one of claims 1 to 6, characterized in that, The xylan has β-(1→4)-D-xylanose as the main chain and the xylan purity is ≥75%.

8. The camellia fruit shell xylan according to claim 7, characterized in that, At a concentration of 2 mg / mL, it inhibits pancreatic lipase by ≥65%.

9. The use of the xylan from Camellia oleifera fruit shell as described in claim 7 or 8 in the preparation of foods, health products, or drugs with weight loss or lipid-lowering functions.

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