A step-by-step extraction of tea saponin coupled with humic acid compound from oil tea dregs and a preparation method and application thereof

Abstract

This invention discloses a stepwise extraction method for tea saponin coupled with humic acid from camellia oil meal, its preparation method, and its application. Tea saponin is extracted from dried camellia oil meal powder using an alcohol solution; the resulting solid mixture is then subjected to alkaline heat treatment to extract humic acid salts; finally, the tea saponin, humic acid salts, and natural active substances are mixed to prepare the tea saponin coupled with humic acid complex extracted from camellia oil meal. This invention fully utilizes camellia oil meal resources, extracts tea saponin and humic acid stepwise, solves the problem of agricultural by-product waste, and couples biomass and artificial humic acid in the process, overcoming the bottleneck of high cost and low value of traditional extraction methods.

Description

Technical Field

[0001] This invention belongs to the field of agricultural solid waste resource utilization and green cosmetics technology, specifically relating to a stepwise extraction method of tea saponin coupled with humic acid complex from camellia seed cake, its preparation method and application. Background Technology

[0002] Camellia oleifera, one of the world's four major woody oil crops, has a long history of cultivation in my country, with its planting scale and camellia oil production ranking first in the world. During camellia oil processing, a large amount of camellia meal byproduct is generated annually, but the current level of resource utilization is low, resulting in a serious waste of biomass resources. Studies have shown that camellia meal is rich in high-value components such as tea saponins, polysaccharides, proteins, and polyphenols. Among them, tea saponins, as a pentacyclic triterpenoid natural surfactant, possess excellent foaming and stabilizing properties, anti-inflammatory activity, and mild detergency, showing significant application potential in the daily chemical industry.

[0003] In the prior art, Chinese patent application CN108383891A discloses a method for co-producing saponins and kaempferol through ethanol extraction-ceramic membrane filtration-resin adsorption. Although this method achieves the separation of the target components, it suffers from drawbacks such as high solvent consumption and high production costs, and it has not overcome the bottleneck of improving the comprehensive value of valuable components. Meanwhile, humic acids, as a class of natural polymers with broad-spectrum biological activity, exhibit excellent performance in anti-inflammatory, skin repair, and sun protection fields. However, the formation cycle of natural humic acids is as long as hundreds of years, limiting their large-scale application. Although CN202411794632.X has achieved rapid preparation of artificial humic acids through a pyrolysis-oxidation process, this technology has not been combined with the co-utilization of agricultural waste and lacks research on functional compounding with natural surfactants such as tea saponins.

[0004] In summary, existing technologies suffer from three major problems: (1) a significant contradiction between the extraction efficiency and economic viability of valuable components from camellia seed meal; (2) a disconnect between the artificial synthesis of humic acid and the synergistic utilization of biomass resources; and (3) a lack of development of the daily chemical efficacy of the tea saponin-humic acid composite system. Based on this, this invention proposes a stepwise extraction technique from camellia seed meal coupled with humic acid preparation. The aim is to construct a composite raw material system with both surface activity and anti-inflammatory functions by extracting high-value components from camellia seed meal in stages, thereby promoting the leapfrog transformation of agricultural and forestry waste into high-value-added daily chemical products. Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a tea saponin-coupled humic acid complex through stepwise extraction from camellia seed cake.

[0006] Another objective of this invention is to provide a tea saponin coupled with humic acid complex obtained by the above preparation method through stepwise extraction of tea camellia seed cake.

[0007] Another object of the present invention is to provide an application of the above-mentioned stepwise extraction of tea saponin coupled with humic acid complex from camellia seed cake.

[0008] This invention enables the efficient utilization of valuable components in camellia seed meal, thus providing green and safe cosmetic raw materials.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing a tea saponin-coupled humic acid complex by stepwise extraction from camellia seed cake, comprising the following steps:

[0011] (1) Mix the dried tea seed cake powder with an ethanol aqueous solution, adjust the pH to 4-12, heat to extract, and obtain mixture 1. Centrifuge to obtain the supernatant and solid mixture.

[0012] (2) The supernatant from step (1) was subjected to vacuum distillation to obtain solid tea saponin; the ethanol aqueous solution was recovered after steam condensation.

[0013] (3) Add the solid mixture from step (1) to the alkaline solution, heat it to obtain mixture 2, centrifuge to obtain a liquid humate aqueous solution and a solid soil conditioner;

[0014] (4) Mix the solid tea saponin from step (2), the aqueous solution of liquid humic acid substances from step (2), the natural active substances and water to obtain the tea saponin coupled humic acid complex extracted stepwise from camellia seed cake.

[0015] Preferably, the dried camellia seed meal powder in step (1) is obtained by washing camellia seed meal, drying it at 40-80℃ to constant weight, and then grinding it through a 40-80 mesh sieve.

[0016] Preferably, the volume percentage of ethanol in the aqueous ethanol solution in step (1) is 10-80%; more preferably, it is 30%.

[0017] Preferably, the mass ratio of the dried camellia seed meal powder and the ethanol aqueous solution in step (1) is 1:1 to 1:20; more preferably, it is 1:8.

[0018] Preferably, the heating extraction temperature in step (1) is 40-90℃, more preferably 70℃; the time is 2-24h, more preferably 4h; and the stirring speed is 50-800rpm, more preferably 200rpm.

[0019] Preferably, the centrifugation separation in step (1) refers to centrifugation at 1000-8000 rpm for 5-30 min, followed by filtration through a filter membrane to obtain a mixture of supernatant and solid.

[0020] More preferably, the pore size of the filter membrane is 0.22 μm.

[0021] Preferably, the reagent used to adjust the pH in step (1) includes at least one of sodium hydroxide and hydrochloric acid.

[0022] Preferably, the pressure of vacuum distillation in step (2) is 20-80 kPa and the temperature is 40-70℃.

[0023] Preferably, the concentration of the alkaline solution in step (3) is 0.5-5 mol / L; more preferably, it is a 1 mol / L sodium hydroxide solution.

[0024] Preferably, the ratio of the solid mixture to the alkaline solution in step (3) is 1g:1-10mL, more preferably the ratio of the solid mixture to the alkaline solution is 1g:3mL.

[0025] Preferably, the temperature of the heat treatment in step (3) is 150-300℃ and the time is 1-3h.

[0026] Preferably, the centrifugation separation in step (3) refers to centrifugation at 1000-8000 rpm for 5-30 minutes, followed by filtration through a filter membrane to obtain a liquid humate aqueous solution and a solid soil conditioner.

[0027] More preferably, the pore size of the filter membrane is 0.22 μm.

[0028] Preferably, in step (4), the solid tea saponin is 0-50% by mass percentage, the liquid humic acid acid aqueous solution is 2-60%, the natural active substance is 3-50% and the water is the balance, wherein the solid tea saponin is not 0.

[0029] Preferably, the aqueous solution of liquid humate in step (4) includes an aqueous solution of sodium humate.

[0030] Preferably, the natural active substance in step (4) includes at least one of the following: Tremella polysaccharide, hydrolyzed wheat protein, yeast β-glucan, trehalose, chitosan and vitamin E.

[0031] More preferably, in step (4), the solid tea saponin is 1±0.2% by mass percentage, the liquid humic acid aqueous solution is 50±2%, the natural active substance is 4±0.5% and the water is the balance.

[0032] Secondly, the present invention provides a tea saponin coupled with humic acid complex obtained by the above preparation method through stepwise extraction of tea meal.

[0033] Thirdly, the present invention provides the application of the above-mentioned stepwise extraction of tea saponin coupled with humic acid complex from tea meal in cosmetics and personal care products.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] (1) Full utilization of camellia seed meal resources, step-by-step extraction of tea saponin and humic acid, solving the problem of waste of agricultural by-products;

[0036] (2) The tea saponin-humic acid complex system works synergistically to enhance antioxidant effects through multiple barriers;

[0037] (3) The process couples biomass and artificial humic acid, breaking through the bottleneck of high cost and low value of traditional extraction. Attached Figure Description

[0038] Figure 1 This is a flowchart of the stepwise extraction process of tea saponins coupled with humic acid from camellia seed cake in Example 1.

[0039] Figure 2 This is the HPLC chromatogram of tea saponin from Example 1.

[0040] Figure 3 This is a scanning electron microscope image of tea saponin from Example 1.

[0041] Figure 4 This is a scanning electron microscope image of sodium humate from Example 1.

[0042] Figure 5 This is a stability test diagram of the tea saponin coupled with humic acid complex extracted from camellia seed cake in Example 1.

[0043] Figure 6 This is a scanning electron microscope image of the soil conditioner from Example 1. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0045] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available products.

[0046] Example 1

[0047] 100g of dried camellia seed cake powder, dried to constant weight at 65℃ and ground through a 60-mesh sieve, was added to 800g of a 30% (v / v) alcohol solution. The pH was adjusted to 10 with NaOH, and the mixture was placed in a 70℃ water bath and extracted at 200 rpm for 4 hours to obtain mixture 1. Mixture 1 was centrifuged at 5000 rpm for 10 minutes for preliminary separation, followed by separation through a 0.22µm membrane to obtain a supernatant and a solid mixture. The supernatant was purified by vacuum distillation at 60 kPa and 60℃ to obtain 8.13g of oven-dried tea saponin. The vapor from the vacuum distillation was condensed and used as a reserve for alcohol. 20g of the oven-dried solid mixture was added to 60 mL of a 1mol / L NaOH solution. The mixture was then hydrothermally heated at 200℃ for 2 hours, followed by centrifugation at 5000 rpm for 10 minutes. The supernatant was separated through a 0.22µm membrane to obtain a liquid sodium humate solution (85.23g / L) and a solid soil conditioner. 1g of oven-dried tea saponin, 50 mL of liquid sodium humate solution, 2g of chitosan, 1g of vitamin E, 1g of D-panthenol, and the remainder water were mixed evenly to obtain 100g of tea saponin coupled with humic acid complex extracted from camellia seed cake.

[0048] Example 2

[0049] 100g of dried camellia seed cake powder, dried to constant weight at 65℃ and ground through a 60-mesh sieve, was added to 800g of a 10% (v / v) alcohol solution. The pH was adjusted to 10 with NaOH, and the mixture was placed in a 70℃ water bath and extracted at 200 rpm for 4 hours to obtain mixture 1. Mixture 1 was centrifuged at 5000 rpm for 10 minutes for preliminary separation, followed by separation through a 0.22µm membrane to obtain a supernatant and a solid mixture. The supernatant was purified by vacuum distillation at 60 kPa and 60℃ to obtain 5.23g of oven-dried tea saponin. The vapor from the vacuum distillation was condensed and used as a reserve for alcohol. 20g of the oven-dried solid mixture was added to 60 mL of a 1mol / L NaOH solution. The mixture was then hydrothermally heated at 200℃ for 2 hours, followed by centrifugation at 5000 rpm for 10 minutes. The supernatant was separated through a 0.22µm membrane to obtain a liquid sodium humate solution (87.32g / L) and a solid soil conditioner. 1g of oven-dried tea saponin, 50 mL of liquid sodium humate solution, 2g of chitosan, 1g of vitamin E, 1g of D-panthenol, and the remainder water were mixed evenly to obtain 100g of tea saponin coupled with humic acid complex extracted from camellia seed cake.

[0050] Example 3

[0051] 100g of dried camellia seed meal powder, dried to constant weight at 65℃ and ground through a 60-mesh sieve, was added to 800g of a 50% ethanol solution. The pH was adjusted to 10 with NaOH, and the mixture was extracted in a 70℃ water bath at 200 rpm for 4 hours to obtain mixture 1. Mixture 1 was centrifuged at 5000 rpm for 10 minutes for preliminary separation, followed by separation through a 0.22µm membrane to obtain a supernatant and a solid mixture. The supernatant was purified by vacuum distillation at 60 kPa and 60℃ to obtain 10.23g of oven-dried tea saponin. The vapor from the vacuum distillation was condensed and used as a reserve for ethanol. 20g of the oven-dried solid mixture was added to 60 mL of a 1mol / L NaOH solution. The mixture was then hydrothermally heated at 200℃ for 2 hours, followed by centrifugation at 5000 rpm for 10 minutes. The supernatant was separated through a 0.22µm membrane to obtain a liquid sodium humate solution (10.45g / L) and a solid soil conditioner. 1g of oven-dried tea saponin, 50 mL of liquid sodium humate solution, 2g of chitosan, 1g of vitamin E, 1g of D-panthenol, and the remainder water were mixed evenly to obtain 100g of tea saponin coupled with humic acid complex extracted from camellia seed cake.

[0052] Example 4

[0053] 100g of dried camellia seed cake powder, dried to constant weight at 65℃ and ground through a 60-mesh sieve, was added to 800g of an 80% alcohol solution. The pH was adjusted to 10 with NaOH, and the mixture was placed in a 70℃ water bath and extracted at 200 rpm for 4 hours to obtain mixture 1. Mixture 1 was centrifuged at 5000 rpm for 10 minutes for preliminary separation, followed by separation through a 0.22µm membrane to obtain a supernatant and a solid mixture. The supernatant was purified by vacuum distillation at 60 kPa and 60℃ to obtain 9.56g of oven-dried tea saponin. The vapor from the vacuum distillation was condensed and used as a reserve for alcohol. 20g of the oven-dried solid mixture was added to 60 mL of a 1mol / L NaOH solution. The mixture was then hydrothermally heated at 200℃ for 2 hours, followed by centrifugation at 5000 rpm for 10 minutes. The supernatant was separated through a 0.22µm membrane to obtain a liquid sodium humate solution (23.19g / L) and a solid soil conditioner. 1g of oven-dried tea saponin, 50 mL of liquid sodium humate solution, 2g of chitosan, 1g of vitamin E, 1g of D-panthenol, and the remainder water were mixed evenly to obtain 100g of tea saponin coupled with humic acid complex extracted from camellia seed cake.

[0054] Comparative Example 1

[0055] Compared with Example 1, the difference is that 50 mL of liquid sodium humate solution, 2 g of chitosan, 1 g of vitamin E, 1 g of D-panthenol, and the remainder of water were mixed evenly to obtain 100 g of humic acid complex extracted from camellia seed cake.

[0056] Comparative Example 2

[0057] Compared with Example 1, the difference is that 1g of oven-dried tea saponin, 2g of chitosan, 1g of vitamin E, 1g of D-panthenol, and the remainder of water are mixed evenly to obtain 100g of tea saponin complex extracted from camellia seed cake.

[0058] Comparative Example 3

[0059] Compared with Example 1, the difference is that 1g of oven-dried tea saponin and the remainder of water are mixed evenly to obtain 100g of tea saponin complex extracted from camellia seed cake.

[0060] Comparative Example 4

[0061] Compared with Example 1, the difference is that 50 mL of liquid sodium humate solution and the remainder of water were mixed evenly to obtain 100 g of humic acid complex extracted from camellia seed cake.

[0062] The scavenging rate of hydroxyl radicals (·OH) by the tea saponin-coupled humic acid complexes extracted from tea seed cakes obtained in Examples 1-4 and the complexes obtained in Comparative Examples 1-4 was tested as follows: 5 mL of a 1% (w / w) complex solution was placed in a 50 mL colorimetric tube. Then, 0.5 mL of 6 mmol / L FeSO4 solution and 0.5 mL of 6 mmol / L H2O2 solution were added, and the mixture was thoroughly mixed. The reaction was carried out at room temperature for 10 min. Next, 0.5 mL of 6 mmol / L salicylic acid solution was added to the reaction system, and the mixture was thoroughly mixed again. The reaction was continued at room temperature for 30 min. After the reaction was completed, the mixture was immediately centrifuged (5000 rpm, 5 min). The absorbance of the supernatant was measured at 510 nm and recorded as A1. Using deionized water as the control group (i.e., replacing 0.5 mL of 6 mmol / L salicylic acid solution with 0.5 mL of deionized water), the reaction and absorbance were measured according to the above steps, and the resulting absorbance was recorded as A0. Anhydrous ethanol was used instead of salicylic acid (i.e., no salicylic acid was added, but an equal amount of anhydrous ethanol was added, with other operations and reagent additions remaining unchanged), and the reaction and absorbance were measured according to the steps, and the resulting absorbance was recorded as A2. The scavenging rate of hydroxyl radicals (·OH) was calculated as: scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%, and the results are shown in Table 1.

[0063] The inhibition rates of the tea saponin-coupled humic acid complex extracted from tea seed cake obtained in Examples 1-4 and the complex obtained in Comparative Examples 1-4 on 5α-reductase were tested using the following methods: The positive control was finasteride with a purity >98%, diluted with anhydrous ethanol to a mass concentration of 0.1%; the complex sample was diluted with pure water to a mass concentration of 1%; and the negative control was PBS solution. During the experiment, sample groups, positive control groups, and negative control groups were established. After adding the sample group, positive control group, and negative control group, and shaking well, HPLC analysis was performed to determine the testosterone content in each tube. The 5α-reductase inhibition rate (%) corresponding to the change in testosterone content in the sample tube was calculated as (1 - change in testosterone content in the sample tube / change in testosterone content in the blank tube) × 100. Data analysis used an independent samples t-test to compare the 5α-reductase inhibition rates of the experimental sample, positive control, and negative control. Statistical analysis was a two-tailed test with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups, and P < 0.05 indicated a significant difference. The results are shown in Table 1.

[0064] Table 1 Test Results

[0065]

[0066] Stability test of tea saponin coupled with humic acid complex extracted from camellia seed cake: The transmittance was measured every 45 seconds at 45℃, 2500 rpm, and 870 nm wavelength using a LUMiSize instrument from LUM GmbH, Germany. A total of 1000 measurements were taken to obtain the product stability curve.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a tea saponin-coupled humic acid complex by stepwise extraction from camellia seed cake, characterized in that, Includes the following steps: (1) Mix the dried tea seed cake powder with an ethanol aqueous solution, adjust the pH to 4-12, heat to extract, and obtain mixture 1. Centrifuge to obtain the supernatant and solid mixture. (2) The supernatant from step (1) was subjected to vacuum distillation to obtain solid tea saponin; the ethanol aqueous solution was recovered after steam condensation. (3) Add the solid mixture from step (1) to the alkaline solution, heat it to obtain mixture 2, centrifuge to obtain a liquid humate aqueous solution and a solid soil conditioner; (4) Mix the solid tea saponin from step (2), the aqueous solution of liquid humic acid substances from step (2), the natural active substances and water to obtain the tea saponin coupled humic acid complex extracted stepwise from camellia seed cake.

2. The preparation method according to claim 1, characterized in that, The ethanol in the aqueous solution described in step (1) has a mass ratio of 10-80%. And / or, the mass ratio of the dried camellia seed meal powder and the ethanol aqueous solution in step (1) is 1:1-1:20; And / or, the heating extraction temperature in step (1) is 40-90℃ and the time is 2-24h.

3. The preparation method according to claim 1 or 2, characterized in that, The concentration of the alkaline solution in step (3) is 0.5-5 mol / L; the alkaline solution is a sodium hydroxide solution; And / or, the ratio of the solid mixture and the alkaline solution in step (3) is 1g: 1-10mL; And / or, the temperature of the heat treatment in step (3) is 150-300℃; the time is 1-3h.

4. The preparation method according to claim 1 or 2, characterized in that, The pressure of vacuum distillation in step (2) is 20-80 kPa and the temperature is 40-70℃.

5. The preparation method according to claim 1 or 2, characterized in that, In step (4), by mass percentage, solid tea saponin is 0-50%, liquid humic acid acid solution is 2-60%, natural active substances are 3-50% and water is the balance, wherein solid tea saponin is not 0. More preferably, in step (4), the solid tea saponin is 1±0.2% by mass percentage, the liquid humic acid aqueous solution is 50±2%, the natural active substance is 4±0.5% and the water is the balance.

6. The preparation method according to claim 1 or 2, characterized in that, The aqueous solution of liquid humate in step (4) includes an aqueous solution of sodium humate; And / or, the natural active substance in step (4) includes at least one of tremella polysaccharide, hydrolyzed wheat protein, yeast β-glucan, trehalose, chitosan and vitamin E.

7. The preparation method according to claim 1 or 2, characterized in that, The reagents used to adjust the pH in step (1) include at least one of sodium hydroxide and hydrochloric acid; And / or, the dried camellia seed meal powder in step (1) is obtained by washing camellia seed meal, drying it at 40-80℃ to constant weight, and then grinding it through a 40-80 mesh sieve.

8. The preparation method according to claim 1 or 2, characterized in that, The centrifugation separation in step (1) refers to centrifuging at 1000-8000 rpm for 5-30 min, followed by filtration through a filter membrane to obtain a mixture of supernatant and solid. The pore size of the filter membrane is 0.22 μm; And / or, the centrifugation separation in step (3) refers to centrifuging at 1000-8000 rpm for 5-30 min, and then filtering through a filter membrane to obtain a liquid humate aqueous solution and a solid soil conditioner; The pore size of the filter membrane is 0.22 μm.

9. A tea saponin-coupled humic acid complex obtained by the preparation method according to any one of claims 1-8 from camellia seed cake.

10. The application of the stepwise extraction of tea saponin coupled with humic acid complex from tea meal as described in claim 9 in cosmetics and personal care products.

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