Method for efficiently separating active matters such as tea saponin from camellia seed meal

Abstract

The invention discloses a method for efficiently separating tea saponin and other active matters from camellia meal, and particularly relates to the technical field of camellia meal active matter purification, the method comprises the following steps: taking camellia meal powder, adding petroleum ether, degreasing, and carrying out suction filtration to obtain solid phase powder; adding ethanol into the solid-phase powder, performing suction filtration after ultrasonic extraction, and freeze-drying filtrate after ethanol removal to obtain tea saponin and flavone crude extract powder; adding methanol and silica gel into the tea saponin and flavone crude extract powder, mixing, carrying out ultrasonic dissolution, carrying out reduced pressure rotary evaporation drying, taking out, grinding, and loading a sample by a dry method; eluting by using eluents with different concentration gradients to obtain an extracting solution; and the contents of tea saponin and flavone are detected through thin layer chromatography or high performance liquid chromatography. According to the method, by optimizing the elution conditions and combining with an efficient detection means, efficient separation of flavone and tea saponin in the oil tea meal is achieved, and a new thought is provided for extraction of target components.

Description

Technical Field

[0001] The present application relates to the technical field of purification of active substances in camellia oil cake, and in particular to a method for efficiently separating active substances such as tea saponin from camellia tea cake. Background Art

[0002] Camellia oil meal, a major byproduct of camellia oil processing, contains a variety of active ingredients, including tea saponins and flavonoids, and holds high development potential. Tea saponins are a natural surfactant with emulsifying, foaming, and antibacterial properties. Flavonoids, due to their anti-inflammatory, antioxidant, and anti-cancer effects, are widely used in medicine and food. However, the current utilization rate of camellia oil meal is low, and traditional extraction methods, with their limited purity, high cost, and complex processes, have hindered its further development. Summary of the Invention

[0003] The present invention provides a method for efficiently separating active substances such as tea saponin from camellia tea cake. This method achieves efficient separation of flavonoids and tea saponin in camellia oil cake by optimizing elution conditions and combining efficient detection means, providing a new idea for the extraction of target components.

[0004] The object of the present invention is achieved through the following technical solutions:

[0005] A method for efficiently separating tea saponin and other active substances from camellia tea cake, the method comprising the following steps:

[0006] S01. Take tea dregs powder, add petroleum ether, and defatting under ultrasonic stirring in a water bath at 60°C for 2 h. After filtration, obtain petroleum ether phase and solid phase powder;

[0007] S02. Add 80% ethanol to the solid phase powder, perform ultrasonic extraction at 40°C for 1 hour, and then filter. The filtrate is subjected to rotary evaporation to remove the ethanol and then freeze-dried in a vacuum to obtain a crude extract powder of tea saponin and flavonoids;

[0008] S03, placing silica gel in a beaker, adding ethyl acetate, shaking and wet-packing the column, and pressing the column to obtain a chromatography column;

[0009] S04. Methanol and silica gel were added to the tea saponin and flavonoid crude extract powders, mixed, ultrasonically dissolved, dried by rotary evaporation under reduced pressure, taken out and ground, and the sample was dry-loaded;

[0010] S05, eluting with different concentration gradient eluents at a flow rate of 1 mL / min, collecting one tube for every 10 mL of eluent to obtain an extract;

[0011] S06. Detecting the contents of tea saponin and flavonoids by thin layer chromatography or high performance liquid chromatography.

[0012] Preferably, in step S01, the material-liquid ratio of tea seed meal powder to petroleum ether is 1:10.

[0013] Preferably, in step S02, the material-liquid ratio of the solid phase powder to ethanol is 1:10.

[0014] Preferably, in step S05, the composition and ratio of the eluent are as follows:

[0015]

[0016] Preferably, in step S06, the thin layer chromatography detection step is:

[0017] Weigh saponin standard and dissolve it in 10 mL of 80% ethanol to prepare a 1 mg / mL saponin standard solution;

[0018] Similarly, prepare 1 mg / mL crude extract and take 1 mL of the oil extract after rotary evaporation;

[0019] Tea saponins and flavonoids were detected by silica gel thin layer chromatography. Oven-activated silica gel thin layer chromatography plates were spotted with standard solution, crude extract, eluent, and oil extract as mobile phases. After drying at room temperature, the plates were developed with vanillin-sulfuric acid colorimetric reagent. The plates were then heated in an oven at 110°C for 4 minutes.

[0020] Observe the color bands of the samples on the thin layer chromatography plate to determine the presence of tea saponins and flavonoids.

[0021] Preferably, the mobile phase is a solution prepared by mixing ethyl acetate: methanol: water = 4:1.4:0.8.

[0022] Preferably, in step S06, the detection is performed by high performance liquid chromatography under the following conditions:

[0023] (1) Tea saponin liquid phase conditions

[0024] A Kromasil 100-5C18 250×4.6mm, 5μm column was used, the mobile phase was acetonitrile and ultrapure water, and the mobile phase gradient was: 0-3min: 5% acetonitrile, 3-8min: 20% acetonitrile, 8-20min: 5% acetonitrile, 20-22min: 5% acetonitrile; flow rate 1.0mL / min; injection volume 10μL; detection wavelength 348nm; column temperature 30℃.

[0025] (2) Flavonoid liquid phase conditions

[0026] A Kromasil 100-5C18 250×4.6mm, 5μm column was used with acetonitrile:methanol = 2:8, elution time 10min; injection volume 10μL; detection wavelength 260nm; column temperature 30℃.

[0027] (3) Preparation of tea saponin standard curve

[0028] Weigh 10 mg of tea saponin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to prepare 0.8, 0.6, 0.4, and 0.2 mg / mL tea saponin solutions. Draw a standard curve with the peak area as the ordinate and the tea saponin mass concentration as the abscissa.

[0029] (4) Preparation of flavonoid standard curve

[0030] Weigh 10 mg of rutin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to make 0.8, 0.6, 0.4, and 0.2 mg / mL rutin solutions. Draw a standard curve with the peak area as the ordinate and the rutin mass concentration as the abscissa.

[0031] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include:

[0032] This study focused on silica gel column chromatography, using defatted camellia oil cake as the raw material. By systematically optimizing the stationary phase and eluent system (ethyl acetate-methanol-water gradient elution), combined with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) dual detection, a highly efficient separation pathway for flavonoids and tea saponins was established. Experiments demonstrated that, under a gradient of ethyl acetate:methanol:water = 4:1:0.8, 1 column volume of elution effectively separated flavonoids, while 2-3 column volumes yielded high-purity tea saponins, achieving significantly better separation efficiency than conventional methods.

[0033] 2. This method achieves efficient separation and purity improvement of flavonoids and tea saponins by optimizing the gradient elution strategy of silica gel column chromatography; at the same time, a multi-technique analytical system (TLC-HPLC-LC-MS / NMR) is constructed to take into account both separation process monitoring and structure confirmation, forming a complete technical closed loop. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A three-dimensional diagram of the simulated spawning site (indoor simulated pool) for producing supermale fry for Pseudosciaena spp.

[0035] Figure 2 It is a schematic diagram of the structure of the egg-laying and egg-collecting device (front view);

[0036] Figure 3 The yield and purity of tea saponin from camellia seed meal by ultrasound-assisted ethanol extraction at different extraction temperatures;

[0037] Figure 4It is the structural diagram of the egg-laying and egg-collecting device (back);

[0038] Figure 5 This is a schematic diagram of the structure of the spawning pond;

[0039] Figure 6 This is a schematic diagram of the spawning pond structure (with a scraper installed at the bottom);

[0040] Figure 7 A three-dimensional diagram of the simulated spawning site for producing supermale fry of the pseudo-female croaker (viewed from above);

[0041] Figure 8 Spot plate results for crude extracts and oil extracts;

[0042] Figure 9 is the standard curve of flavonoid standards;

[0043] Figure 10 This is the standard curve of tea saponin standard. DETAILED DESCRIPTION

[0044] The following will describe the implementation methods of this application in detail with reference to the accompanying drawings and examples, so that the application can fully understand how technical means are used to solve technical problems and achieve corresponding technical effects, and implement them accordingly. The embodiments of this application and the various features therein can be combined with each other without conflict, and the resulting technical solutions are all within the scope of protection of this application.

[0045] It should be clear that the embodiments described below are only some of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative work are within the scope of protection of this application.

[0046] Example 1:

[0047] This embodiment provides a method for efficiently separating active substances such as tea saponin from camellia tea cake. The specific steps are as follows:

[0048] 1. Materials and instruments are shown in Tables 1 and 2.

[0049] Table 1 Information on the main reagents used in the experiment

[0050]

[0051] Table 2 Information of main instruments used in the experiment

[0052]

[0053]

[0054] 2. Tea saponin and flavonoid extraction process

[0055] Taking the crude extract yield of tea saponin as an indicator, four factors, namely ethanol volume fraction, solid-liquid ratio, extraction temperature and extraction time, were selected for single factor experiment to explore the influence of each factor on the extraction rate of tea saponin. The process levels of each factor are shown in Table 3, and a response surface experiment was conducted.

[0056] Table 3 Factor levels of single factor experiment

[0057]

[0058] 3. Extraction of tea saponins and flavonoids

[0059] Weigh 30g of tea dregs powder and, based on the results of a single-factor level test, add 300mL of petroleum ether at a solid-liquid ratio of 1:10. Degrease the mixture by ultrasonic stirring in a water bath at 60°C for 2h. Filter to obtain a petroleum ether phase and a solid phase powder. Add 20g of the solid phase powder to 200mL of 80% ethanol at a solid-liquid ratio of 1:10. Ultrasonic extraction is performed at 40°C for 1h, followed by filtration. The filtrate is rotary evaporated to remove ethanol and freeze-dried in a vacuum to obtain a crude extract of tea saponins and flavonoids. The petroleum ether phase is rotary evaporated and stored for later use.

[0060] 4. Tea saponin and flavonoid purification process

[0061] Weigh 45g of silica gel into a beaker, add a suitable amount of ethyl acetate, shake well, and wet-pack the column. Press the column. Weigh 1g of the lyophilized crude extract powder, dissolve it in 7mL of methanol, mix with 1g of silica gel, and sonicate. Dry the mixture by rotary evaporation under reduced pressure, remove, and grind. Apply the sample by dry loading. Elute using gradient eluents of varying concentrations. The eluent compositions and ratios are shown in Table 4. The flow rate is 1mL / min. Collect one tube per 10mL of eluent. Rapidly analyze the sample components by thin-layer chromatography and compare the separation performance of the eluents.

[0062] Table 4 Composition and ratio of eluent

[0063]

[0064] 5. Detection of tea saponins and flavonoids

[0065] Thin layer chromatography

[0066] Weigh 10 mg of saponin standard and dissolve it in 10 mL of 80% ethanol to prepare a 1 mg / mL saponin standard solution. Similarly, prepare a 1 mg / mL crude extract and measure 1 mL of the rotary evaporated oil extract.

[0067] Tea saponins and flavonoids were detected using silica gel thin-layer chromatography. Oven-activated silica gel thin-layer chromatography plates were spotted with standard solution, crude extract, eluent, and oil extract. The plates were developed using a mobile phase of (ethyl acetate:methanol:water = 4:1.4:0.8). After drying at room temperature, the plates were developed with vanillin-sulfuric acid as a color developer. The plates were then heated in an oven at 110°C for 4 minutes. The color bands on the plates were observed to preliminarily determine the presence of tea saponins, flavonoids, and other substances.

[0068] High-performance liquid chromatography

[0069] (1) Tea saponin liquid phase conditions

[0070] A Kromasil 100-5C18 (250×4.6mm, 5μm) chromatographic column was used, the mobile phase was acetonitrile and ultrapure water (containing 0.1% formic acid), and the mobile phase gradient was: 0-3min: 5% acetonitrile, 3-8min: 20% acetonitrile, 8-20min: 5% acetonitrile, 20-22min: 5% acetonitrile; flow rate 1.0mL / min; injection volume 10μL; detection wavelength 348nm; column temperature 30℃.

[0071] (2) Flavonoid liquid phase conditions

[0072] A Kromasil 100-5C18 (250×4.6mm, 5μm) column was used with acetonitrile:methanol (formic acid water) = 2:8, elution time 10min; injection volume 10μL; detection wavelength 260nm; column temperature 30℃.

[0073] (3) Preparation of tea saponin standard curve

[0074] Weigh 10 mg of tea saponin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to prepare 0.8, 0.6, 0.4, and 0.2 mg / mL tea saponin solutions. Draw a standard curve with the peak area as the ordinate and the tea saponin mass concentration as the abscissa.

[0075] Preparation of flavonoid standard curve

[0076] Weigh 10 mg of rutin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to make 0.8, 0.6, 0.4, and 0.2 mg / mL rutin solutions. Draw a standard curve with the peak area as the ordinate and the rutin mass concentration as the abscissa.

[0077] Example 2: Structural Identification of Tea Saponins and Flavonoids

[0078] Liquid chromatography-mass spectrometry analysis

[0079] The eluted samples were subjected to spot plate detection. Pure tea saponin and flavonoid test tubes were combined and the solvent was removed by rotary evaporation. After reconstitution with methanol, the samples were prepared in liquid phase vials for liquid chromatography-mass spectrometry analysis. The liquid chromatography was performed using a BEH C18 column (2.1×100 mm, 1.7 μm) and a photodiode array detector (WATERS ACQUITY PDA). The detection wavelength was 200-400 nm, the column temperature was 45°C, and the mobile phase was acetonitrile and an aqueous solution containing 0.1% formic acid. The tea saponin mobile phase concentration gradient was: 10% acetonitrile 90% formic acid in water (0-2 min); 30% acetonitrile 70% formic acid in water (3-5 min); 80% acetonitrile 20% formic acid in water (6-8 min); 100% acetonitrile 90% formic acid in water (9-11 min); and 10% acetonitrile 90% formic acid in water (12-14 min). The flavonoid mobile phase concentration gradient was 20% acetonitrile 80% formic acid in water (8 min). The flow rate was 0.3 mL / min and the injection volume was 5 μL.

[0080] Mass spectrometry was performed using ESI as the ionization source, capillary voltage 3500 V, cone voltage 30 V, ion source temperature 100°C, desolvation temperature 400°C, mass range 20-1500 m / z, and anion and cation detection. The LC / MS data were compared and analyzed using UNIFI software.

[0081] Nuclear magnetic resonance analysis

[0082] The crystal samples of pure flavonoids and tea saponin samples were respectively placed in 25 mL eggplant-shaped flasks and the solvent was removed by rotary evaporation to obtain 20 mg of pure products, which were dissolved in DMSO-d6 (containing internal standard TMS) and transferred to a nuclear magnetic resonance tube for scanning using a nuclear magnetic resonance instrument to measure the 1HNMR and 13CNMR of tea saponin and flavonoids.

[0083] Example 2: Single factor test:

[0084] 1. Effect of material-liquid ratio on the yield and purity of tea saponin crude extract

[0085] The yield and purity of tea saponin from tea seed meal with ultrasonic-assisted ethanol extraction at different material-liquid ratios of tea seed meal and ethanol are shown in Figure 1 .

[0086] When extracting tea saponins from camellia seed meal with ethanol, the solid-liquid ratio has a significant effect on the extraction effect. Studies have shown that when the solid-liquid ratio is in the range of 1:6 to 1:10 g / mL, the yield and purity of the crude extract of tea saponins both show an upward trend with increasing ethanol dosage. This is mainly because the increase in the amount of solvent is conducive to the diffusion and dissolution of tea saponins in camellia seed meal into ethanol, thereby improving the extraction efficiency. However, when the solid-liquid ratio exceeds 1:10 g / mL, the yield and purity of tea saponins tend to stabilize. This may be because most of the tea saponins have been dissolved, and further increasing the amount of solvent has limited improvement on the extraction effect. Therefore, based on the results of the single-factor experiment, the optimal solid-liquid ratio of camellia seed meal to 80% ethanol is 1:10 g / mL.

[0087] 2. Effect of ethanol percentage on the yield and purity of tea saponin crude extract

[0088] The yield and purity of tea saponin from camellia seed meal were obtained by ultrasonic assisted ethanol extraction with different ethanol percentages. Figure 2 .

[0089] Within the ethanol concentration range of 40% to 70%, the extraction yield and purity of the crude tea saponin extract both increased with increasing ethanol concentration. However, when the ethanol concentration exceeded 70%, the extraction yield decreased, while the purity slowly increased. This phenomenon may be because, when the ethanol concentration is below 70%, an appropriate amount of water facilitates the dissolution of tea saponins in the camellia seed meal, but it also increases the solubility of water-soluble substances such as crude protein, starch, polysaccharides, and pigments. This results in an increased extraction yield but relatively low purity of the crude extract. Conversely, high ethanol concentrations may cause flocculation and precipitation of crude protein and polysaccharides, thereby increasing the extraction purity of tea saponins. However, high ethanol concentrations also reduce the solubility of tea saponins, resulting in a decrease in the extraction yield of the crude extract.

[0090] 3. Effect of extraction temperature on the yield and purity of tea saponin crude extract

[0091] The yield and purity of tea saponin from camellia seed cake extracted by ultrasound-assisted ethanol at different extraction temperatures are shown in Figure 3 .

[0092] During the tea saponin extraction process, extraction temperature significantly affects the yield and purity of the final product. Studies have shown that when the extraction temperature is between 40°C and 60°C, the yield and purity of the crude tea saponin extract increase with increasing temperature. However, once the extraction temperature exceeds 60°C, both indicators show a downward trend.

[0093] This phenomenon may be related to changes in the properties of ethanol at high temperatures. High temperatures intensify the movement of ethanol molecules, accelerating their volatilization (ethanol's boiling point is approximately 78°C), thus affecting extraction efficiency. Furthermore, the proteins and polysaccharides in camellia seed meal may undergo thermal denaturation and precipitation, which could also hinder the precipitation of tea saponins.

[0094] Therefore, taking into account both yield and purity, the optimal extraction temperature condition for the single-factor experiment was determined to be 60°C

[40] . This conclusion provides an important reference for optimizing the extraction process of tea saponin, which helps to achieve higher product quality and yield in actual production. Understanding the effect of temperature on the extraction process can help to better control and optimize the extraction process of tea saponin, improve resource utilization and product value.

[0095] 4. Effect of extraction time on the yield and purity of tea saponin crude extract

[0096] The yield and purity of tea saponins from tea seed cakes extracted with ultrasound-assisted ethanol at different extraction times are shown in Figure 4 .

[0097] from Figure 4 It can be seen that as the extraction time gradually increases, the yield of tea saponin crude extract and the purity of tea saponin show an increasing trend, especially within 120 minutes, the rate of increase is relatively large, and after 150 minutes, it tends to be stable. This may be because the extraction of tea saponin from tea seed cake requires infiltration, penetration, desorption, dissolution, diffusion and other processes, so the extraction requires a certain amount of time

[41] . However, as the extraction time increases, the starch and oil substances in the extract are hydrolyzed, so the impurities increase. Therefore, the extraction time of 150 minutes is the optimal level condition for the single factor experiment. In the process of extracting tea saponin, it is necessary to consider the effect of extraction time on the yield and purity. Within a certain period of time, increasing the extraction time can improve the yield and purity of tea saponin, but too long an extraction time may lead to an increase in impurities. Therefore, it is necessary to find an optimal extraction time to obtain tea saponin with a higher yield and purity.

[0098] Example 3: Response surface experiment:

[0099] Based on the analysis of single-factor experimental results, three key process parameters were identified: extraction time (A), ethanol percentage (B), and extraction temperature (C). Under a fixed solid-liquid ratio (1:10 g / mL), the yield of the tea saponin crude extract (Y) was selected as the optimization metric. A four-factor, three-level experimental design was constructed using response surface methodology (RSM). The specific parameter settings are shown in Table 5. Multiple regression analysis of the experimental data was performed using Design Expert 13.0.1.0 software, and a mathematical model was established between the yield and process parameters.

[0100] Table 5 Box-Behnken design table and effect values

[0101]

[0102]

[0103] By further evaluating the significance of the three variables (extraction time, ethanol percentage, and extraction temperature) on the crude tea saponin yield, a quadratic polynomial model was optimized. A total of 17 experiments were conducted, all of which were repeated three times. The quadratic polynomial model showed the correlation between extraction time, ethanol percentage, extraction temperature, and crude tea saponin yield. Through regression analysis of the data results, the quadratic polynomial obtained is as follows:

[0104] Y=30.93-0.0313A-0.3350B+0.3662C-0.0925AB+0.2250AC+0.5625BC-1.92A2-1.57B2-1.93C2

[0105] Y: tea saponin crude extraction rate; A: extraction time; B: ethanol percentage; C: extraction temperature

[0106] The coefficient of determination (R²) was 0.9334, demonstrating that the model had good predictability and fully explained the relationships between the variables. The p-value for the response surface model was 0.0024, which was less than 0.05, indicating that the model was statistically significant and that the predictor variables in the model had a significant effect on the response variable. A signal-to-noise ratio greater than 4 was considered satisfactory. The model had a ratio of 8.7085, indicating a sufficient signal.

[0107] The relationship between the three variables is shown by a three-dimensional response surface plot and a contour plot ( Figure 5 The relationship between the crude tea saponin yield and various variables was clarified, facilitating the determination of optimal reaction conditions for tea saponin extraction from camellia oil seed meal. Based on the optimized polynomial model, a crude tea saponin yield of 30.161% was predicted under the conditions of an extraction time of 169.354 minutes, an ethanol percentage of 68.953%, and an extraction temperature of 60.590°C. Parallel validation experiments revealed an actual crude tea saponin yield of 29.48% ± 1.23%, a purity of 78.34%, and a tea saponin yield of 23.09%.

[0108] Verification test:

[0109] To facilitate practical application, the optimal extraction conditions were revised to: 69% ethanol concentration, 60°C extraction temperature, and 170 minutes extraction time. Three parallel experiments were conducted under these revised conditions, and the crude tea saponin yield was measured to be 29.48% ± 1.23%, with a relative standard deviation of 2.46%. The relative error from the predicted theoretical value was 2.25%, less than 5%. This indicates that the regression model fits the actual situation well and the experimental results are reliable.

[0110] Response surface analysis interaction:

[0111] The slope of the response surface diagram and the shape of the contour line directly reflect the magnitude of the interaction between the factors. The Box-Behnken module of Design Expert 13.0.1.0 software was used to draw the 3D response surface diagram of the interaction in the above regression equation model ( Figure 5 ).from Figure 5 It can be seen that in the effect of the two-factor interaction term on the yield of tea saponin crude extract, the interaction between the curves of ethanol percentage (B) and extraction temperature (C) is significant, the response surface is convex and steep, and the projection is nearly elliptical, while the interaction between other factors is not significant.

[0112] Example 4: Extraction of tea saponins and flavonoids

[0113] The mass of the freeze-dried crude extract powder was weighed to calculate the crude extract yield. The tea saponin and flavonoid contents were determined by HPLC to calculate the purity.

[0114]

[0115] Example 5: Tea saponin and flavonoid purification process

[0116] Silica gel was wet-loaded with ethyl acetate and then dry-loaded to compare the elution effects of different eluents. This experiment employed different concentration gradients using a two-component system (methanol:ethyl acetate) and a three-component system (ethyl acetate:methanol:water). Five elution runs were performed, with 10 mL of eluent collected in a test tube and then spot-blotted until no tea saponin remained. The elution results are shown in Table 6. Tubes containing the same composition were combined, dried, reconstituted with methanol, and then spot-blotted to verify the composition. Pure flavonoids with only a single band and crystals from pure tea saponin were screened for analysis by liquid chromatography-mass spectrometry and nuclear magnetic resonance.

[0117] Table 5 Elution results of different eluents

[0118]

[0119]

[0120] As can be seen from Table 5, adding component water to the developing solvent can significantly improve the developing effect and improve the tailing of the component. The reason may be that water is a polar substance with greater polarity than methanol, which increases the polarity of the eluent, thereby improving the elution effect. In addition, the silanol (-SiOH) on the surface of the silica gel easily forms hydrogen bonds with the components. Adding water can form hydrogen bonds with the silanol, thereby reducing its interaction with the components and better eluting from the silica gel column, thereby improving the tailing.

[0121] The 4th and 5th elutions had better results. The elution point plate results are shown in Figure 6 and Figure 7 .

[0122] Example 6: Detection of tea saponins and flavonoids

[0123] Thin layer chromatography:

[0124] (1) Selection of developing agent ratio

[0125] Because the silica particles in thin-layer silica gel plates are finer and have a surface area approximately twice that of a chromatographic column, a developing solvent system with an Rf value of approximately 0.3 for sample separation on thin-layer plates is optimal for column chromatography. Specific developing solvent ratios and Rf values are shown in Table 6.

[0126] Table 6 Expanding system selection

[0127]

[0128] The final developing agent system selected was ethyl acetate:methanol:water = 4:1:0.3 with an Rf value of 0.3 for tea saponin and 4:1.4:0.8 with an Rf value of 0.3 for flavonoids. This was the best theoretical developing agent for the gradient development.

[0129] (2) Selection of color developer ratio

[0130] Since the color development effect of the color developer is different for different substances, the color developer ratio needs to be adjusted to achieve the best color development effect for the target substance. The color developer ratio is adjusted. The color developer ratio and color development effect are shown in Table 7.

[0131] Table 7 Color developer ratio selection

[0132]

[0133] Finally, 2g vanillin + 20mL anhydrous ethanol + 0.2mL concentrated sulfuric acid was selected as the color developer ratio for subsequent experiments. However, the vanillin-sulfuric acid color developer is unstable and prone to oxidation, decomposition and other reactions, so it needs to be prepared before use.

[0134] (3) Testing of standards and samples

[0135] After preparing the standard solution and sample, prepare the developing solvent with ethyl acetate: methanol: water = 4:1.4:0.8, and prepare the vanillin-sulfuric acid color developer with 2 g vanillin + 20 mL anhydrous ethanol + 0.2 mL concentrated sulfuric acid.

[0136] Spot-dry an oven-activated silica gel TLC plate, develop it, and air dry it at room temperature. Irradiate the developed plate with 254nm and 365nm UV light. Under 254nm UV light, the oil appears gray and hazy, while the flavonoids appear as dark black spots. Tea saponins show no color. Under 365nm UV light, the oil fluoresces, the flavonoids appear as dark purple spots, and tea saponins show no color. Using a color developer and heating at 110°C for 4 minutes, the oil appears red, the flavonoids appear yellow, and the tea saponins appear purple.

[0137] Saponin standard, tea saponin, rutin standard, crude extract, oil extract spot plate results see Figure 8 , which can provide a control for the subsequent elution sample detection. The results of this plate spotting show that rutin and the flavonoid components in the sample are not the same substance.

[0138] High-performance liquid chromatography:

[0139] The standard curves of flavonoids and tea saponins are as follows Figure 9 、 Figure 10 shown.

[0140] Based on the thin-layer chromatography results, the pure tea saponin and flavonoids obtained in the fourth and fifth separations were subjected to liquid chromatography-mass spectrometry. Each sample was tested three times and the average peak area was taken. The peak area of the flavonoids in the fourth separation at a reconstitution concentration of 0.3 mg / mL was 4,687,804 μv.s⁻¹, with a calculated purity of 86.03%. The peak area of the flavonoids in the fifth separation at a reconstitution concentration of 0.3 mg / mL was 4,704,899 μv.s⁻¹, with a calculated purity of 86.36%. The peak area of the tea saponin in the fourth separation at a reconstitution concentration of 0.45 mg / mL was 294,812 μv.s⁻¹, with a calculated purity of 90.67%. The peak area of the flavonoids in the fifth separation at a reconstitution concentration of 0.45 mg / mL was 311,780 μv.s⁻¹, with a calculated purity of 96.43%.

[0141] The analysis results show that the elution conditions of the fifth silica gel column have better separation effect, and the pure flavonoids contain some impurities. The combined spot plate results show that the flavonoid extract contains impurities such as oil; the purity of the substance separated from tea saponin is relatively pure, and it contains a small amount of impurities that may be polysaccharides.

[0142] Conclusion: This study successfully achieved efficient, stepwise separation of flavonoids and tea saponins from camellia oilseed meal by optimizing silica gel column chromatography gradient elution conditions (ethyl acetate:methanol:water = 4:1:0.8). Experimental results demonstrated that flavonoids were preferentially separated at 1 column volume, with a purity of 86.36%. High-purity tea saponins, reaching 96.43%, were obtained after 2-3 column volumes of elution. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis confirmed the separation efficiency and structural integrity of the target components. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) were further used to determine the molecular weight, functional groups, and stereochemistry of the polysaccharides in the flavonoids and tea saponins, providing a scientific basis for the precise identification of natural products.

[0143] The innovation of this study lies in the construction of a "TLC-HPLC-LC-MS / NMR" multi-technique coupling system, forming a complete technical closed loop from separation process monitoring to structure confirmation. Compared with traditional methods, silica gel column chromatography combined with gradient elution significantly improved the separation efficiency, and it is easy to operate and cost-effective. The research results not only provide new ideas for the large-scale extraction of active ingredients from camellia oleifera meal, but also lay a technical foundation for its application in the fields of medicine, daily chemicals, etc. However, there are still a small amount of impurities (such as oils and polysaccharides) in the pure flavonoids, and the elution conditions need to be further optimized or a secondary purification process needs to be introduced; in addition, the large-scale application of silica gel columns needs to solve the problems of solvent recovery and cost control.

[0144] Overall, this study has promoted the high-value utilization of camellia oilseed meal resources through technological innovation, providing important technical support for the expansion of mountain economic chains and the enhancement of the added value of the camellia oilseed industry. Future research could combine green solvents (such as supercritical CO2) with high-efficiency separation techniques (such as molecular imprinting) to further enhance the environmental and economic efficiency of the process. Furthermore, further research on the structure-activity relationship between flavonoids and tea saponins could be conducted to expand their potential applications in functional materials and precision medicine.

Claims

1. A method for efficiently separating active substances such as tea saponin from camellia tea cake, characterized in that: The steps include: S01. Take tea seed meal powder, add petroleum ether, and defatting in a water bath with ultrasonic stirring at 60 °C for 2 h. After filtration, obtain petroleum ether phase and solid phase powder; S02, adding 80% ethanol to the solid phase powder, ultrasonically extracting at 40°C for 1 h, and then filtering, the filtrate was subjected to rotary evaporation to remove ethanol, and then freeze-dried in a vacuum to obtain tea saponin and flavonoid crude extract powder; S03, placing silica gel in a beaker, adding ethyl acetate, shaking and wet-packing the column, and pressing the column to obtain a chromatography column; S04. Methanol and silica gel were added to the tea saponin and flavonoid crude extract powders, mixed, ultrasonically dissolved, dried by rotary evaporation under reduced pressure, taken out and ground, and the sample was dry-loaded; S05. Elute with gradient eluents of different concentrations at a flow rate of 1 mL / min, collecting one tube for every 10 mL of eluent to obtain an extract; S06. Detecting the contents of tea saponin and flavonoids by thin layer chromatography or high performance liquid chromatography.

2. The method according to claim 1, characterized in that In step S01, the material-liquid ratio of tea seed powder and petroleum ether is 1:

10.

3. The method according to claim 1, characterized in that In step S02, the material-liquid ratio of the solid phase powder and ethanol is 1:

10.

4. The method according to claim 1, wherein In step S05, the composition and ratio of the eluent are as follows: 。 5. The method according to claim 1, wherein In step S06, the thin layer chromatography detection steps are: Weigh saponin standard and dissolve it in 10 mL of 80% ethanol to prepare a 1 mg / mL saponin standard solution; Similarly, prepare 1 mg / mL crude extract and take 1 mL of the oil extract after rotary evaporation; Tea saponins and flavonoids were detected by silica gel thin layer chromatography. Oven-activated silica gel thin layer chromatography plates were spotted with standard solution, crude extract, eluent, and oil extract as mobile phases. After drying at room temperature, the plates were developed with vanillin-sulfuric acid colorimetric reagent. The plates were then heated in an oven at 110°C for 4 minutes. Observe the color bands of the samples on the thin layer chromatography plate to determine the presence of tea saponins and flavonoids.

6. The method according to claim 5, characterized in that The mobile phase was a solution prepared by mixing ethyl acetate: methanol: water = 4:1.4:0.

8.

7. The method according to claim 1, characterized in that In step S06, the product is obtained by high performance liquid chromatography under the following conditions: (1) Tea saponin liquid phase conditions A Kromasil 100-5C18 250 × 4.6 mm, 5 μm column was used with acetonitrile and ultrapure water as the mobile phase. The mobile phase gradient was as follows: 0-3 min: 5% acetonitrile, 3-8 min: 20% acetonitrile, 8-20 min: 5% acetonitrile, 20-22 min: 5% acetonitrile; the flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 348 nm; and the column temperature was 30 °C.

8. (2) Flavonoids liquid phase conditions A Kromasil 100-5C18 250 × 4.6 mm, 5 μm column was used with acetonitrile:methanol = 2:8, elution time 10 min, injection volume 10 μL, detection wavelength 260 nm, and column temperature 30 °C.

9. (3) Preparation of tea saponin standard curve Weigh 10 mg of tea saponin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to prepare 0.8, 0.6, 0.4, and 0.2 mg / mL tea saponin solutions. Draw a standard curve with the peak area as the ordinate and the tea saponin mass concentration as the abscissa.

10. (4) Preparation of flavonoid standard curve Weigh 10 mg of rutin standard into a 10 mL volumetric flask and prepare a 1 mg / mL stock solution with 80% ethanol. Take a certain volume of the stock solution and dilute it with 80% ethanol to make 0.8, 0.6, 0.4, and 0.2 mg / mL rutin solutions. Draw a standard curve with the peak area as the ordinate and the rutin mass concentration as the abscissa.

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