Preparation method of plant extract based on plasma activated water coupled pulsed electric field

Abstract

The invention belongs to the technical field of plant extraction, and provides a preparation method of a plant extract based on a plasma activated water coupled pulsed electric field. The preparation method comprises the following steps: mixing plasma activated water and plants to obtain slurry, then performing pulsed electric field treatment, and finally performing solid-liquid separation to obtain the plant extract. According to the method, irreversible electroporation is instantaneously formed on a plant cell membrane by using a pulsed electric field, meanwhile, plasma activated water with high reaction activity is used as an extraction solvent, active particles rich in the plasma activated water can rapidly permeate through the electroporation, osmotic pressure inside and outside cells and solubility of components are changed, and under the synergistic interaction of physical wall breaking and chemical permeation promotion, the plant cell membrane can be rapidly extracted. The target active ingredients (such as flavone and anthraquinone) can be efficiently'extracted 'in an extremely short time (reaching the second level), so that the extraction efficiency is greatly improved by adopting the method disclosed by the invention. And the adopted raw materials and reagents are green, environment-friendly and high in safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, and more particularly to a preparation method of plant extract based on plasma-activated water coupled with pulsed electric field. BACKGROUND

[0002] The active ingredients of Hemerocallis citrina Baroni flower, such as flavonoids and anthraquinones (e.g., emodin), mainly exist in specific organelles within cells. Traditional hot extraction or organic solvent extraction methods not only have low efficiency, but also are difficult to fully destroy the tough cell wall. More seriously, long-term high-temperature (e.g., higher than 60℃) treatment can significantly accelerate the oxidative degradation of anthraquinones such as emodin, resulting in deepening of the color of the extract, reduction of biological activity, and possible production of unnecessary by-products. Therefore, the traditional extraction technology of Hemerocallis citrina Baroni flower (such as water extraction and alcohol extraction) has obvious bottlenecks, as follows: (1) contradiction between efficiency and activity: high temperature and long time extraction process can improve the yield, but it is easy to cause the oxidation, degradation or inactivation of heat-sensitive active ingredients (such as part of flavonoids and vitamins), reducing the biological potency of the final product; (2) solvent residue and environmental pressure: the use of organic solvents (such as methanol and acetone) is beneficial to improve the extraction efficiency, but there are safety risks of flammability and explosion, solvent residue problems, and do not meet the requirements of green and sustainable modern industrial development; (3) poor selectivity: the traditional method extracts a complex mixture, and the purity of the target active ingredient is low, and the subsequent separation and purification steps are complicated and costly.

[0003] Therefore, it is urgent to develop a method for efficiently, quickly, greenly and selectively extracting active ingredients in plants (such as Hemerocallis citrina Baroni flower). SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a preparation method of plant extract based on plasma-activated water coupled with pulsed electric field. The extraction method of the present application has high extraction efficiency (the extraction rate of total flavonoids and polyphenols of Hemerocallis citrina Baroni flower can reach 1.28-2.85% and 1.07-2.42%, respectively), fast extraction speed (from "hour level" to "second level", achieving nearly instantaneous extraction), high selectivity, high retention degree of active ingredients of the obtained extract, and the raw reagent used is green and safe.

[0005] The first aspect of the present application provides a preparation method of plant extract based on plasma-activated water coupled with pulsed electric field.

[0006] Specifically, the preparation method of plant extract based on plasma-activated water coupled with pulsed electric field comprises the following steps: (1) taking plasma-activated water; (2) mixing the plant and the plasma-activated water to obtain a slurry; (3) subjecting the slurry to pulsed electric field treatment to obtain treated slurry; (4) subjecting the treated slurry to solid-liquid separation, and collecting the liquid to obtain the plant extract.

[0007] The plant and the plasma-activated water (PAW) are mixed, and then the pulsed electric field is used to form irreversible electroporation on the plant cell membrane instantaneously to create a channel for extraction. Meanwhile, the plasma-activated water with high reactivity in the system is used as an extraction solvent, and the active particles contained in the plasma-activated water can rapidly penetrate the electroporation, change the osmotic pressure inside and outside the cell, and change the solubility of the components, so that the target active ingredients (such as flavonoids and anthraquinones) can be efficiently extracted in a very short time (which can reach seconds). Therefore, the method of the present application realizes the synergistic effect of physical wall breaking and chemical penetration promotion, and greatly improves the efficiency of extracting active substances from plants. In addition, the active particles (such as •OH and H2O2) contained in the plasma-activated water are electrophilic reagents with high reactivity, which preferentially interact instantaneously and strongly with functional groups such as flavonoid phenolic hydroxyl groups and anthraquinone conjugated systems. This interaction can effectively weaken the non-covalent bond force between the target active ingredients and the internal structure of the cell, significantly improve the mass transfer rate and solubility from solid to liquid, and thus realize targeted “release promotion” and improve the selectivity of active ingredients.

[0008] Preferably, in step (1), the oxidation-reduction potential of the plasma-activated water is +250 mV to +450 mV, and / or the pH value of the plasma-activated water is 2.0-4.0. The plasma-activated water has high oxidation activity and acidity, and the pH 2.0-4.0 is the best dissolution pH window for flavonoids and anthraquinone components, which further promotes the selective dissolution. At the same time, the environment with high oxidation-reduction potential can instantaneously inactivate the oxidase (such as polyphenol oxidase) in the cell, which fundamentally avoids the enzymatic oxidation degradation of the target components during the extraction process, and selectively retains the natural active conformation of the active ingredients. The plasma-activated water can improve the extraction efficiency, protect the active ingredients, improve the retention rate and color of the active ingredients, and is conducive to realizing low-temperature, instantaneous, efficient and green extraction.

[0009] Preferably, in step (1), the plasma-activated water is prepared by a preparation method comprising the following steps: placing water in a plasma generator to generate the plasma-activated water under normal pressure plasma jet.

[0010] Preferably, the water is deionized water or ultrapure water.

[0011] Preferably, the working gas of the plasma generator is inert gas and / or nitrogen, and / or the flow rate of the working gas of the plasma generator is 2-4 SLM, and / or the power of the plasma generator is 50-150 W.

[0012] Preferably, the inert gas is at least one of argon, helium, neon.

[0013] Preferably, the plasma generator is further provided with a water treatment reactor, and the water treatment reactor contains a cooling jacket.

[0014] Preferably, the water temperature of the plasma-activated water is 10-30℃. The cooling jacket of the water treatment reactor can maintain the water temperature at 10-30℃. In the process of extracting by using the plasma-activated water, the lower temperature can avoid the damage of active ingredients.

[0015] Preferably, the treatment time of the water in the plasma generator is 3-15 minutes.

[0016] Preferably, the plasma-activated water is used within 10-20 minutes after preparation. Using fresh plasma-activated water for extraction can ensure the extraction effect.

[0017] Preferably, in step (2), the plant is pretreated before being mixed with the plasma-activated water, and the pretreatment is vacuum freeze-drying of the plant, and then crushing to obtain a powder.

[0018] Preferably, the average particle size of the powder is 40-80 mesh.

[0019] Preferably, in step (2), the plant is at least one of daylily flower, rose flower, chrysanthemum, honeysuckle, tea leaf, mulberry leaf, licorice, and turmeric.

[0020] Preferably, in step (2), the solid-liquid ratio of the plant to the plasma-activated water is 1:10-1:50 g / mL.

[0021] Preferably, in step (3), the electric field strength of the pulse electric field is 10-35 kV / cm; and / or the pulse width of the pulse electric field is 5-100 μs; and / or the pulse number of the pulse electric field is 5-100.

[0022] Preferably, in step (3), the flow rate of the slurry through the pulse electric field is 50-200 mL / min, and / or the temperature of the slurry is ≤45℃. The lower temperature of the plant during the treatment by the pulse electric field can avoid the damage of active ingredients.

[0023] Preferably, in step (4), the plant extract contains flavonoid active ingredients and / or phenolic acid active ingredients. Further preferably, in step (4), the plant extract contains flavonoid active ingredients and phenolic acid active ingredients.

[0024] Preferably, in step (4), the mass of the flavonoid active ingredients accounts for 1-1.5% of the total mass of the plant extract.

[0025] Preferably, in step (4), after the collection of the liquid, drying is performed to obtain a powder.

[0026] Preferably, the drying is spray drying.

[0027] Compared with the prior art, the present application has the following advantages: The present application mixes plasma-activated water and plants to obtain a slurry, then performs pulsed electric field treatment, and finally obtains a plant extract after solid-liquid separation. The present application utilizes pulsed electric field to form irreversible electroporation on the plant cell membrane. At this time, the plasma-activated water with high reactivity in the system acts as an extraction solvent, and the active particles contained therein can rapidly penetrate through the electroporation, change the osmotic pressure and solubility of the components inside and outside the cell, and under the synergistic effect of physical wall breaking and chemical penetration promotion, the target active ingredients (such as flavonoids and anthraquinones) can be efficiently "extracted" in a very short time (which can reach seconds), and the selectivity is high. Therefore, the method of the present application greatly improves the efficiency of extracting active ingredients from plants. Moreover, the raw reagent used is green and environmentally friendly, and has high safety. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A real object diagram of the Hemerocallis fulva powder used in Example 1 of the present application; Figure 2 A comparison diagram of the chicken embryo stimulation test results of Example 1, the positive control group and the negative control group of the present application. DETAILED DESCRIPTION

[0029] In order to make those skilled in the art more clearly understand the technical solutions described in the present application, the following examples are provided for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0030] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0031] Example 1 A preparation method of a plant extract based on plasma-activated water coupled with pulsed electric field, comprising the following steps: (1) Preparation of PAW: Deionized water was placed in a plasma generator to generate plasma-activated water under atmospheric pressure plasma jet. The argon flow rate was 3 SLM, the power was 80W, the deionized water treatment time was 5 minutes, and the water temperature in the water treatment reactor (containing a cooling jacket) was controlled at 20℃. The ORP of the obtained PAW was +320mV and the pH was 2.8. The plasma-activated water was used within 10-20 minutes after preparation. (2) Mixing: Add 10g of daylily pollen (such as...) Figure 1 As shown, daylily pollen is a white powder. It is mixed with 250 mL of freshly prepared PAW (material-to-liquid ratio 1:25 g / mL) to obtain a slurry. (3) Extraction: The slurry was passed through the pulsed electric field (PEF) treatment chamber at a flow rate of 100 mL / min, with an electric field strength of 25 kV / cm, a pulse width of 20 μs, and 20 pulses to obtain the treated slurry; (4) Separation: The treated slurry was immediately centrifuged at 8000 rpm for 10 minutes, the supernatant was collected, and spray-dried to obtain daylily flower extract (powder state).

[0032] Example 2 A method for preparing plant extract based on plasma-activated water coupled pulsed electric field differs from Example 1 in that, in step (1), the treatment time of deionized water is extended to 8 minutes, and the ORP of the obtained PAW is +380 mV and pH=2.5; in step (3), the PEF electric field strength is increased to 30 kV / cm.

[0033] Example 3 A method for preparing plant extract based on plasma-activated water coupled pulsed electric field differs from Example 1 in that, in step (2), 10g of daylily pollen is mixed with 200mL of freshly prepared PAW (material-liquid ratio 1:20 g / mL).

[0034] Example 4 A method for preparing plant extract based on plasma-activated water coupled pulsed electric field differs from Example 1 in that, in step (3), the slurry is passed through the PEF treatment chamber at a flow rate of 100 mL / min, the electric field strength is set to 15 kV / cm, the pulse width is 20 μs, and the number of pulses is 20.

[0035] Example 5 A method for preparing plant extract based on plasma-activated water coupled pulsed electric field differs from Example 1 in that, in step (3), the slurry is passed through the PEF treatment chamber at a flow rate of 100 mL / min, the electric field strength is set to 25 kV / cm, the pulse width is 20 μs, and the number of pulses is 10.

[0036] Example 6 A method for preparing plant extract based on plasma activated water coupled with pulsed electric field, which is different from example 1 in that, in step (1), the flow rate of nitrogen is 3 SLM, the power is 80 W, and the deionized water is treated for 5 minutes, with the water temperature controlled at 20°C. The ORP of the prepared PAW is +290 mV, and the pH is 3.1.

[0037] Example 7 A method for preparing plant extract based on plasma activated water coupled with pulsed electric field, which is different from example 1 in that, in step (3), the slurry is passed through the PEF treatment chamber at a flow rate of 50 mL / min (reduced flow rate, extended action time in the PEF treatment chamber), the electric field strength is set to 25 kV / cm, the pulse width is 20 μs, and the number of pulses is 20.

[0038] Example 8 A method for preparing plant extract based on plasma activated water coupled with pulsed electric field, comprising the following steps: (1) Preparation of PAW: argon flow rate 3 SLM, power 80 W, treatment of deionized water for 8 minutes, water temperature controlled at 20°C, the prepared PAW ORP is +380 mV, pH=2.5.

[0039] (2) Mixing: 10g of Hemerocallis fulva powder is mixed with 250mL of freshly prepared PAW (solid-liquid ratio 1:25 g / mL) to obtain a slurry; (3) Extraction: the slurry is passed through the PEF treatment chamber at a flow rate of 100 mL / min, the electric field strength is set to 20 kV / cm, the pulse width is 20 μs, and the number of pulses is 30, to obtain the treated slurry; (4) Separation: the treated slurry is immediately centrifuged at 8000 rpm for 10 minutes, the supernatant is collected, and after spray drying, Hemerocallis fulva flower extract (powder state) is obtained.

[0040] Example 9 A method for preparing plant extract based on plasma activated water coupled with pulsed electric field, which is different from example 1 in that, the Hemerocallis fulva powder in step (2) is replaced by an equal weight of honeysuckle.

[0041] Comparative Example 1 (traditional hot water extraction method) A method for preparing plant extract, comprising the following steps: Take 10g of Hemerocallis fulva powder, add 250mL of deionized water, and reflux extract in a 90°C water bath for 2 hours, and centrifuge to obtain the supernatant.

[0042] Comparative Example 2 (single ethanol extraction method) A method for preparing a plant extract, comprising the following steps: Take 10 g of Hemerocallis fulva pollen, add 250 mL of 70% ethanol solution, shake at room temperature for 4 hours, centrifuge to take the supernatant, and recover ethanol by rotary evaporation.

[0043] Comparative Example 3 (only PEF + deionized water) A method for preparing a plant extract, which is different from Example 1 in that PAW is replaced by an equal weight of deionized water.

[0044] Comparative Example 4 (only PAW, no PEF treatment) A method for preparing a plant extract, comprising the following steps: (1) Preparation of PAW: argon flow rate 3 SLM, power 80 W, treatment of deionized water for 5 minutes, water temperature control at 20°C, the prepared PAW ORP is +320 mV, pH=2.8.

[0045] (2) Mixing: 10 g of Hemerocallis fulva pollen is mixed with 250 mL of freshly prepared PAW (solid-liquid ratio 1:25 g / mL) to obtain a slurry; (3) Extraction: magnetic stirring at room temperature for 30 minutes, then centrifugation to obtain Hemerocallis fulva flower extract.

[0046] Comparative Example 5 (ultrasonic-assisted water extraction) A method for preparing a plant extract, comprising the following steps: Take 10 g of pollen, add 250 mL of deionized water, ultrasonic extraction at 40°C and 400 W power for 30 minutes, and centrifuge.

[0047] Product effect test 1. Hemerocallis fulva flower flavonoid and polyphenol mass percentage content test Total flavonoid test method and principle: Rutin is used as the most representative substance in flavonoids, and its structure meets the basic structure standard of flavonoids, i.e. the conjugated system of A ring and C ring. Since the ultraviolet detector has a very obvious absorption to this conjugated system, the use of rutin as a control can effectively determine the content of total flavonoids. Based on the fact that rutin and aluminum ions form a stable complex under acidic conditions. Specifically, rutin reacts with aluminum ions to form a complex, which has a characteristic absorption peak under ultraviolet-visible spectrophotometer. By measuring the absorption peak of the complex in the sample, the content of total flavonoids in the sample can be indirectly determined.

[0048] (1) Test steps Preparation of standard curve: Accurately weigh 20 mg of rutin, dissolve it in 30% ethanol and dilute to 100 mL, containing 0.2 mg of rutin per mL. Take 10 clean test tubes, add different volumes of rutin standard solution in turn, supplement to 5 mL with 30% ethanol by volume, add sodium nitrite and aluminum nitrate solution, and finally add sodium hydroxide. After mixing and standing for 10 minutes, measure the absorbance (OD) at 510 nm, and draw the standard curve.

[0049] Sample determination: Take an appropriate amount of sample solution and follow the standard curve preparation procedure to measure the absorbance. Calculate the total flavonoid content in the sample according to the standard curve, using the formula (1) as follows: Total flavonoid content = 983.4 x OD x dilution factor ÷ sample amount Formula (1) Total polyphenol content test method and principle: The principle of Folin phenol method for determining polyphenol content is based on the oxidation-reduction reaction between Folin reagent (Folin-Ciocalteu reagent) and polyphenols. Folin reagent is a phenolic substance with reducing properties, which can react with polyphenols under alkaline conditions to form blue, purple or red complexes with high absorbance. The color depth of this complex is positively correlated with the content of polyphenols, so the content of polyphenols can be calculated by measuring the absorbance of the reaction solution.

[0050] Operation method: Reagent preparation: Dilute the Folin reagent and prepare a standard polyphenol solution with appropriate concentration. To ensure the accuracy and reproducibility of the experiment, the reagents should be prepared before the experiment and stored in the dark.

[0051] Reaction: After mixing the extract with Folin reagent, add an alkaline solution (such as sodium hydroxide solution) and measure the absorbance at a specific wavelength (765 nm) using a spectrophotometer after sufficient reaction. Record the sample absorbance value and compare it with the standard curve to calculate the polyphenol content in the sample.

[0052] Determination of absorbance: Use a spectrophotometer or other equipment to measure the absorbance of the reaction solution.

[0053] Calculation: According to the absorbance value of the reaction solution, refer to the standard curve to calculate the content of polyphenols in the sample to be tested.

[0054] The test results are as follows.

[0055] Table 1 Total flavonoid and polyphenol content of each example and comparative sample (% w / w)

[0056] From the above table, compared with Comparative Examples 1-5, the extraction rates of total flavonoids and polyphenols of Hemerocallis fulva flowers in Inventive Examples 1-8 can reach 1.28-2.85% and 1.07-2.42% respectively, and the extraction rate of active ingredients is high. Among them, in Inventive Examples 1-8, the extraction rates of total flavonoids and polyphenols of Hemerocallis fulva flowers in Inventive Example 1 are the highest, and the mass concentrations are 1.37% and 1.16% respectively.

[0057] Comparative Example 1 (hot water extraction) high temperature extraction leads to serious degradation of polyphenols. Comparative Example 2 (ethanol extraction) solvent properties affect selectivity, resulting in a decrease in polyphenol content. Comparative Example 3 (PEF + water) only has physical wall breaking, lacks chemical penetration promotion, has the lowest efficiency, and also has a lower polyphenol content. Comparative Example 4 (PAW soaking) only has chemical penetration promotion, lacks physical wall breaking, still has insufficient efficiency, and also has a lower polyphenol content. Comparative Example 5 (ultrasonic water extraction) physical shearing is effective, but the thermal effect and oxidation cause serious loss of polyphenols.

[0058] Honeysuckle itself is a plant rich in flavonoids and polyphenols, and its reference content is usually higher than that of Hemerocallis fulva flowers.

[0059] 2. In vitro test of anti-glycation (1) Test model: BSA-glucose / fructose model Test principle: In vitro simulation of glycosylation reaction, incubate bovine serum albumin (BSA) with reducing sugar (such as glucose) at high temperature (37℃), after adding the sample to be tested, evaluate the anti-glycation activity by detecting the generated AGEs or fluorescence product.

[0060] Test steps: Incubation system: BSA (10 mg / mL) + glucose (0.5 mol / L) + sample to be tested (different test concentrations). Conditions: constant temperature incubation at 37℃ for 7 days (simulation of long-term glycation) or accelerated reaction at high temperature (50℃ constant temperature incubation for 24 hours). Detection by fluorescence method: determination of OD 395 absorbance value, AGEs have spontaneous fluorescence characteristics (excitation wavelength 370 nm, emission wavelength 440 nm).

[0061] Calculation: Anti-glycation rate (%) = [1-(treated group AGEs content / control group AGEs content)] x 100%.

[0062] Among them, the control group contains only sugar and protein without the sample to be tested; the treated group contains sugar, protein and the sample to be tested.

[0063] Test concentration of sample to be tested: The extracts (powder state) prepared in the above examples and comparative examples are diluted with water to obtain sample aqueous solutions with mass fraction concentrations of 0.5wt%, 1wt% and 2wt%, and then the efficacy test is carried out.

[0064] (2) Test results Table 2 Anti-glycation rate (%) of samples of each example and comparative example

[0065] As shown in the above table, the anti-glycation rates of Examples 1-8 are higher than those of Comparative Examples 1-5 at different concentrations. Among Examples 1-8, the anti-glycation rate of Example 1 is the highest at each concentration, which proves that the extract is rich in complete and highly active functional ingredients, and the anti-glycation rates at concentrations of 0.5wt%, 1wt% and 2wt% are 14.31%, 25.63% and 31.17% respectively, which are concentration-dependent.

[0066] Comparative Example 3 has the weakest efficacy because the use of deionized water as a solvent results in incomplete extraction of active ingredients and insufficient total amount of functional ingredients. The efficacy of Comparative Example 4 (PAW soaking) is better than that of Comparative Example 3 but not as good as that of Example 1, which indicates that although the components in the extract of Comparative Example 4 are less damaged, the extract fails to efficiently “extract” all the functional ingredients in the cells, which again confirms the necessity of synergistic effect. The active ingredient content in Comparative Examples 1, 2 and 5 is low, and the anti-glycation rate is also low accordingly.

[0067] The results of Examples 1-8 and Example 9 show that the extracted honeysuckle extract contains more abundant and more active anti-glycation ingredients (such as chlorogenic acid and flavonoids).

[0068] 3. ABTS free radical scavenging rate test Test principle: The ABTS free radical scavenging capacity detection method is an in vitro detection method for evaluating the ABTS free radical scavenging capacity of antioxidant samples.

[0069] Test steps: (1) Reagent preparation: Mix 7.4 mmol / L ABTS solution, 2.6 mmol / L potassium persulfate solution and PBS phosphate buffer (pH 7.3, solvent) at a volume fraction of 1:1:40 to prepare a working solution, and wait for reaction for 12 h in the dark at room temperature.

[0070] (2) Sample treatment: Dilute the above working solution with ethanol by 40-50 times.

[0071] (3) Determination of absorbance: Mix the working solution with the sample solution (0.5wt%, 1wt% and 2wt% sample aqueous solution) to obtain a mixed solution, and determine the absorbance of the mixed solution before and after reaction.

[0072] (4) Data processing: Calculate the ABTS free radical scavenging rate of the sample according to the change in absorbance.

[0073] Evaluation method: Calculate the ABTS free radical scavenging rate of each sample at the concentration of 0.5wt%, 1wt% and 2wt% respectively to evaluate their antioxidant activity. Generally speaking, the higher the ABTS free radical scavenging rate value, the stronger the antioxidant activity of the sample.

[0074] Table 3 ABTS free radical scavenging rate (%) of samples of each example and comparative example

[0075] From the above table, the ABTS free radical scavenging rates of examples 1-8 are all higher than those of comparative examples 1-5, and the ABTS free radical scavenging capacity is highly related to the content of polyphenol and other antioxidant substances, indicating that the antioxidant substances in plants can be well extracted by the present application. Among them, the ABTS free radical scavenging rate of example 1 is the highest, indicating that the effect of scavenging ABTS free radicals is the best, and the ABTS free radical scavenging rates at different concentrations of 0.5wt%, 1wt% and 2wt% are 34.51%, 60.73% and 89.15% respectively.

[0076] The effect of comparative example 4 (PAW soaking) is better than that of comparative example 3 (PEF+water), which is due to the protective effect of the acidic environment of PAW on phenolic substances and its own certain extraction capacity for polyphenols. However, the effect of example 1 is still superior to that of comparative examples 3 and 4, which indicates that the coupling of PEF and PAW can most effectively "transport" the endogenous antioxidant substances in cells and keep them in a high active state in the extraction liquid.

[0077] 4, Inhibition of AGER gene expression in HDF-α human dermal cells (qPCR test) Basic principle: HDF-α cells are treated by siRNA or inhibitor to down-regulate AGER gene expression, and the inhibition effect is verified by qPCR.

[0078] Operation method: Cell culture and treatment. Cell recovery and subculture: HDF-α cells are cultured in DMEM medium containing 10wt% fetal bovine serum (FBS), and cultured at 37℃, 5% CO2. When the cell density reaches 80%-90%, subculture (0.25% trypsin digestion).

[0079] siRNA transfection (take 24-well plate as an example): Grouping: Experimental group: AGER siRNA (target sequence needs to be verified).

[0080] Negative control group: Non-targeting siRNA.

[0081] Blank control group: only transfection reagent (Mock).

[0082] Step: Dilute siRNA: Mix siRNA with Opti-MEM (final concentration 20-50 nmol / L) according to the instruction. Prepare transfection complex: Mix siRNA with Lipofectamine 3000 at a volume ratio of 1:1, stand at room temperature for 15 minutes. Cell seeding: Seed HDF-a cells into 24-well plate at a density of 5 x 10 4 / well, and incubate for 24 hours. Transfection: Remove old medium, add transfection complex, and replace with complete medium after incubation at 37°C for 6 hours. Continue to incubate for 48-72 hours (optimized according to the action time of siRNA).

[0083] RNA extraction: Remove medium, wash cells with PBS twice. Add Trizol (500 μL per well), lyse cells, and transfer to EP tube. Extract total RNA according to the Trizol method or kit instructions. RNA quality detection: Determine RNA concentration using Nanodrop (A260 / A280 should be between 1.8 and 2.0). Verify RNA integrity by agarose gel electrophoresis (28S / 18S rRNA ratio ≈ 2:1).

[0084] cDNA synthesis: Reverse transcription reaction: Take 1 μg of total RNA and perform reverse transcription according to the kit instructions (such as PrimeScript RT Master Mix). Reaction conditions: 37°C / 15 minutes (reverse transcription), 85°C / 5 seconds (enzyme inactivation). Store the product at -20°C for later use.

[0085] qPCR detection of AGER expression: Primer design: AGER (NM_001136): Forward primer: 5'-CAGGACCACCAGCAGTATCA-3' Reverse primer: 5'-GCAGCAGCATCTTCATCACC-3' Data analysis: Ct value acquisition: The qPCR instrument automatically generates Ct value (threshold cycle number).

[0086] Relative expression calculation: Use 2^(-ΔΔCt) method: Calculate ΔCt = Ct(AGER) - Ct(GAPDH).

[0087] Calculate ΔΔCt = ΔCt (experimental group) - ΔCt (control group).

[0088] Relative expression = 2^(-ΔΔCt).

[0089] Inhibition rate calculation: AGER inhibition rate (%) = (1 Control group relative expression amount / experimental group relative expression amount) x 100% Test concentration: The test cytotoxicity of 1000 ppm (0.1%, w / w) is almost non-toxic to HDF-a human dermal fibroblast cells (cell survival rate > 90%), so the test concentration is set to 0.1% (w / w).

[0090] Calculate the inhibition rate: (1 - relative expression of the sample group to the gene) / 1 * 100% Table 4 AGER inhibition rate test results of samples of each example and comparative example

[0091] Among them, the AGER inhibition rate of the blank control group is 100%.

[0092] From the above table, examples 1-8 are all higher than comparative examples 1-5, wherein at a concentration of 0.1% (w / w), example 1 has the best inhibition efficiency of AGER gene mRNA relative expression, which is 20.34%. The efficacy test results at the cell level show that the extract of the application not only has a high content of chemical components, but also has a better preservation of complex and synergistic efficacy component spectrum by the extraction method of the application.

[0093] The inhibition rates of comparative example 3 and comparative example 4 are lower than that of example 1, which proves that the extract obtained by a single technical path has a lower biological titer than that obtained by a synergistic technical path, which again confirms the superiority of the coupled extraction method of the application from the "function" level.

[0094] 5. Eye irritation test Test item: Test the eye irritation / corrosion of plant extract Test basis: SN / T 2329-2009 "Cosmetic Eye Irritation / Corrosion Chicken Embryo Chorioallantoic Membrane Test" Operation steps: CAM (chick embryo chorioallantoic membrane) preparation: purchase 0-day-old chicken embryos, incubate to 9-day-old, check and discard defective chicken embryos. The 9-day-old chicken embryos are subjected to egg inspection, and the air chamber position is marked on the surface of the eggshell; the marked part of the eggshell is removed with a serrated curved forceps to expose the white egg membrane, and the operation should be careful not to damage the integrity of the egg membrane. A few milliliters of 0.9% sodium chloride solution are added dropwise to wet the egg membrane, and the inner membrane is removed with forceps to ensure that the blood vessel membrane is not damaged.

[0095] Irritation scoring method: At least 6 chicken embryos in each group, 0.3 mL of the test substance is directly added to the surface of the CAM, the reaction of the CAM is observed, and the time of each toxic effect within 5 min is recorded. Including three reactions of hemorrhage, blood vessel lysis and blood clotting.

[0096] End point scoring method: At least 6 chicken embryos per group, 0.3 mL of the test substance was directly applied to the CAM, ensuring that at least 50% of the CAM surface was covered by the test substance. After 3 min of application, the test substance on the CAM membrane was gently washed with normal saline, and the results were observed after 30 s of washing. The degree of change in the three toxic effects of hemorrhage, vasolysis and coagulation was observed. If the observation showed that the score of at least one reaction of all 6 chicken embryos was moderate or above (total score ≥ 12), the test should be repeated once.

[0097] The negative control group and the positive control group should be tested by irritation scoring method.

[0098] Negative control group: 0.9% sodium chloride (NaCl) solution; positive control group: 0.1 mol / L sodium hydroxide solution.

[0099] Since each sample is a clear solution, it is suitable for irritation scoring method (IS).

[0100] Irritation scoring method: the irritation score (IS) is calculated by using the following formula (2), and the result is kept to two decimal places: Formula (2) In the formula, SecH, SecL, SecC represent the average time of observing the beginning of hemorrhage, vasolysis and coagulation on the CAM membrane, respectively, in seconds (s).

[0101] Table 5: Evaluation criteria for irritation scoring method results

[0102] Test acceptance criteria:

[0103] Sample test concentration: 2% (w / w) aqueous sample solution.

[0104] Test results: Table 6: Test results of chicken embryos of each example and comparative sample

[0105] As can be seen from the above table, all the samples of the examples and the comparative examples at a mass fraction concentration of 2% are non-irritating to chicken embryos, indicating that the ethanol-water system and the extraction process have little effect on the irritation of chicken embryos.

[0106] All the samples of the examples and the comparative examples have no eye irritation at a concentration of 2%. This indicates that the specific extraction process used in the present application has no direct correlation with irritation. The extraction process of the present application is green and environmentally friendly, does not use organic solvents, and ensures that the product has good safety.

[0107] For example, Figure 2As shown in A-B, after the extract of Example 1 of the present application was used to treat the chick embryo for 300s, the chick embryo had no obvious change and was still consistent with that at 0s, indicating that the extract had little irritation. As shown in Figure 2 As shown in C-D, after the chick embryo was treated with 0.1 mol / L NaOH aqueous solution (positive control group) for 300s, the blood vessels of the chick embryo ruptured, indicating that the NaOH aqueous solution had great irritation. As shown in Figure 2 As shown in E-F, after the chick embryo was treated with 0.9% NaCl aqueous solution (negative control group) for 300s, the chick embryo had no obvious change and had no irritation.

[0108] According to the above results, for the daylily flower, active ingredients such as flavonoids and anthraquinones (such as emodin) mainly exist in the parenchyma tissue or specific organelles with dense cell wall structure, and the extraction is difficult. In the present application, the plasma activated water coupled with the pulsed electric field technology is applied to the extraction of the daylily flower. The pulsed electric field can instantaneously break the cell membrane in nanoseconds-microseconds to form reversible or irreversible electroporation, which creates an excellent channel for the penetration of PAW. The high oxidation-reduction potential and acidic environment of PAW can effectively enhance the solubility and diffusion rate of the target components, and the short action time can avoid the hydrolysis reaction caused by long-term acid soaking. This synergistic mode of “physical opening and chemical rapid extraction” perfectly overcomes the two long-standing problems of difficult cell wall breaking and easy degradation of heat-sensitive active ingredients (which generally deteriorate at a temperature higher than 60°C), and realizes the efficient and high-quality extraction of active ingredients from the daylily flower under low-temperature and instantaneous conditions, as well as the high-selectivity extraction of flavonoids, anthraquinones and other specific active ingredients.

[0109] The extraction method of the present application has at least the following advantages: (1) Short extraction time: the extraction time can reach “seconds”, realizing nearly instantaneous extraction and improving the production efficiency by orders of magnitude. Through the synergistic effect of physical wall breaking by PEF and chemical penetration promotion by PAW, the extraction yield of key active ingredients (such as total flavonoids and emodin) of the daylily flower is significantly higher than that of any single traditional method (hot water extraction, alcohol extraction) or single advanced physical field method (ultrasound, single PEF).

[0110] (2) Low extraction temperature: the temperature of the entire extraction process is always lower than 45°C, which maximally preserves the chemical structure and biological activity of heat-sensitive active ingredients and ensures the high efficiency of the final product.

[0111] (3) High product purity and stability: the acidic environment of PAW helps to stabilize phenolic compounds, and the instantaneous extraction reduces the time window for oxidative degradation, resulting in better color and purity of the obtained extract.

[0112] (4) Green and safe: using water and air / inert gas as precursors, completely abandoning toxic, flammable organic solvents, fundamentally solving the problems of solvent residue and safety hazards. No toxic and harmful waste is discharged in the production process, and PAW can be naturally degraded, meeting the requirements of green environmental protection and sustainable development.

[0113] (5) Low energy consumption and low cost potential: PEF technology itself has low energy consumption, although PAW preparation requires energy, but the extremely short extraction time brings the optimization of overall energy consumption. Avoid the cost of purchasing, recycling, processing of organic solvents, and the investment of related safety protection facilities. From the whole life cycle, it has significant cost advantage.

[0114] (6) Simple and controllable process flow: high degree of system modularization, easy to realize continuous and automatic production, in line with the development direction of modern industrial intelligent manufacturing, and also easy to control the stability and consistency of product quality between different production batches, conducive to standardized production.

Claims

1. A method for preparing a plant extract, characterized in that, Includes the following steps: (1) Take plasma-activated water; (2) The plant and the plasma-activated water are mixed to obtain a slurry; (3) The slurry is subjected to pulsed electric field treatment to obtain the treated slurry; (4) After solid-liquid separation of the treated slurry, the liquid is collected to obtain the plant extract.

2. The method for preparing the plant extract according to claim 1, characterized in that, In step (1), the oxidation-reduction potential of the plasma-activated water is +250mV to +450mV, and / or the pH value of the plasma-activated water is 2.0-4.

0.

3. The method for preparing the plant extract according to claim 1, characterized in that, In step (1), the plasma-activated water is prepared by a method including the following steps: water is placed in a plasma generator and plasma-activated water is generated under atmospheric pressure plasma jet.

4. The method for preparing the plant extract according to claim 3, characterized in that, The working gas of the plasma generator is an inert gas and / or nitrogen, and / or the flow rate of the working gas of the plasma generator is 2-4 SLM, and / or the power of the plasma generator is 50-150W.

5. The method for preparing the plant extract according to claim 1, characterized in that, In step (2), the temperature of the plasma-activated water is 10-30℃.

6. The method for preparing the plant extract according to claim 1, characterized in that, In step (2), the plant is at least one of daylily, rose, chrysanthemum, honeysuckle, tea, mulberry leaf, licorice, and turmeric.

7. The method for preparing the plant extract according to claim 1, characterized in that, In step (2), the solid-liquid ratio of the plant and the plasma-activated water is 1:10-1:50 g / mL.

8. The method for preparing the plant extract according to claim 1, characterized in that, In step (3), the electric field strength of the pulsed electric field is 10-35 kV / cm; and / or, the pulse width of the pulsed electric field is 5-100 μs; and / or, the number of pulses of the pulsed electric field is 5-100.

9. The method for preparing the plant extract according to claim 1, characterized in that, In step (3), the flow rate of the slurry through the pulsed electric field is 50-200 mL / min, and / or the temperature of the slurry is ≤45℃.

10. The method for preparing the plant extract according to claim 1, characterized in that, In step (4), the plant extract contains flavonoid active ingredients and / or phenolic acid active ingredients.

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