Method for extracting and preparing high-purity aloe-emodin from aloe leaf epidermis in one step

Abstract

The invention discloses a method for extracting and preparing high-purity aloe-emodin from aloe leaf epidermis in one step. The method comprises the following steps: pretreating aloe leaf epidermis; mixing choline chloride and lactic acid, heating and stirring until the solution is clear and transparent, cooling, and adding pure water to obtain a eutectic solvent; mixing the treated aloe leaf epidermis with a deep-eutectic solvent to obtain a raw material solution; putting the raw material solution into pulsed electric field equipment for treatment; and centrifugally separating an object obtained by pulsed electric field treatment, adding water into a liquid phase after separation, refrigerating to obtain a precipitate, filtering, separating, washing and freeze-drying the precipitate to obtain the high-purity aloe-emodin. The method is simple in step, low in energy consumption and high in efficiency, no toxic organic solution is needed in the preparation process, no acid-base waste liquid is generated, ferric chloride is not adopted, no wastewater containing ferrous iron is generated, and the method is free of pollution, environmentally friendly and low in preparation cost; and the obtained aloe-emodin is high in purity and strong in free radical scavenging capacity.

Description

Technical Field

[0001] The present invention relates to the field of the preparation of aloe-emodin, and particularly relates to a method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis. Background Art

[0002] Aloe-emodin is an anthraquinone compound containing multiple phenolic hydroxyl groups and has various active functions, such as anti-pathogenic microorganisms, antioxidant, anti-inflammatory, anti-tumor, antiviral, regulating the body's immune function, conditioning the skin, and protecting the cardiovascular system, etc. Aloe-emodin is widely used in industries such as medicine, health care, and cosmetics due to its excellent biological activity. Usually, the activity of aloe-emodin is positively correlated with its purity. Therefore, how to efficiently and low-costly prepare high-purity aloe-emodin is of great significance for the industrial application of aloe-emodin.

[0003] At present, the methods for preparing aloe-emodin are relatively cumbersome, and acidic or alkaline toxic waste liquids will be generated during the preparation process, with low efficiency and high cost. For example, Chinese invention patent CN202310736289.2 discloses a method for preparing aloe-emodin. In this method, aloin is extracted from aloe paste and prepared into aloe-emodin. A 3-6% hydrochloric acid solution is used to extract from the raw material for 20 minutes to obtain a crude extract of aloin; hydrochloric acid triethylamine-ferric chloride ionic liquid is added for reaction to convert aloin into aloe-emodin (heat preservation and reflux for 8 hours); toluene is used for reflux extraction for 8 hours for purification, and only after cooling and crystallization can high-purity aloe-emodin be obtained. A large amount of hydrochloric acid solution, ferric chloride aqueous solution, and toluene are required in the whole reaction process of this technology, and more toxic waste liquids will be generated subsequently.

[0004] Chinese Patent Application CN202310951446.1 discloses a method for extracting aloe-emodin from rhubarb, which requires four steps of enzymatic hydrolysis, extraction, alkaline alcohol dissolution, and acid precipitation to obtain aloe-emodin. The specific steps are as follows: 1) Add an enzyme (any one or a combination of cellulase, glucoamylase, rhamnase, pectinase) accounting for 0.1%-0.5% of the mass of the rhubarb raw material and carry out enzymatic hydrolysis at 30-50°C for 2-5 hours to obtain an enzymolyzed material; 2) Add an organic solvent with a volume 3-10 times that of the rhubarb raw material to the enzymolyzed material and carry out extraction at 30-90°C, and concentrate the extract to a specific gravity of the extract paste of 1.05-1.20; 3) Add a methanol aqueous solution or an ethanol aqueous solution with a mass 20-50 times that of the extract paste and a mass concentration of 80%-90% to the extract paste, and then add an alkaline solution accounting for 20%-30% of the mass of the extract paste and dissolve for 2-5 hours; 4) Place the filtrate at room temperature, adjust the pH to 3-5 with acid, and let it stand for sedimentation for 0.5-2 hours. This method not only inevitably requires the use of expensive enzymes and generates acid-base waste liquids during the preparation process, but also the steps are relatively cumbersome and time-consuming. It takes at least more than 5 hours to extract and finally prepare aloe-emodin from the raw materials.

[0005] In view of the disadvantages of the existing methods for preparing aloe-emodin, such as cumbersome steps, generation of toxic waste liquids, high energy consumption, and high costs, there is an urgent need in the art for a method that is simple, environmentally friendly, and low-cost to prepare high-purity aloe-emodin. Summary of the Invention

[0006] In order to overcome the deficiencies and defects in the prior art, the purpose of the present invention is to provide a method for directly extracting and preparing high-purity aloe-emodin from the aloe leaf epidermis, realizing the preparation of high-purity aloe-emodin with high efficiency, low cost, and environmental friendliness.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for directly extracting and preparing high-purity aloe-emodin from aloe skin, which is characterized by including the following steps:

[0009] 1) Raw material preparation: Pretreat the aloe leaf epidermis;

[0010] 2) Preparation of deep eutectic solvent: Mix choline chloride and lactic acid, heat and stir until the solution is clear and transparent, and add pure water after cooling to obtain a deep eutectic solvent;

[0011] 3) Preparation of raw material solution: Mix the pretreated aloe leaf epidermis with the deep eutectic solvent to obtain a raw material solution;

[0012] 4) Pulsed electric field treatment: Place the raw material solution in a pulsed electric field device for treatment;

[0013] 5) Separation: Centrifuge the product obtained by pulsed electric field treatment, add water to the liquid phase after separation, refrigerate to obtain a precipitate, filter and separate the precipitate, wash it, and then lyophilize it to obtain high-purity aloe-emodin.

[0014] To better achieve the object of the present invention, preferably, in step 1), the pretreatment is to wash the aloe leaf epidermis, remove the surface dirt, and cut it into small pieces for later use.

[0015] Preferably, the aloe leaf epidermis is the epidermis of fresh aloe arborescens leaves; the small pieces cut are 7×7 mm - 11×11 mm small pieces.

[0016] Preferably, in step 2), the molar ratio of choline chloride to lactic acid is (1:1)-(1:5);

[0017] The heating and stirring is carried out by stirring in a water bath at a temperature of 85 - 95°C and a rotation speed of 200 - 400 rpm; the heating and stirring time is 1 - 2 hours.

[0018] Preferably, in step 2), the mass percentage concentration of the deep eutectic solvent is 70% - 80%.

[0019] Preferably, in step 3), taking grams and milliliters as units, the mass ratio of the aloe leaf epidermis to the volume of the deep eutectic solvent is (1:3)-(1:10).

[0020] Preferably, in step 4), the treatment time of the pulsed electric field equipment is 10 - 20 min, the electric field frequency is 150 - 200 Hz, and the excitation voltage is 95 - 125 V.

[0021] Preferably, in step 5), the centrifugal force for centrifugal separation is 5000 - 10000 rpm, and the volume of water added is 4 - 8 times the volume of the liquid after centrifugal separation;

[0022] The temperature of refrigeration is 1 - 7°C, the refrigeration time is 1 - 5 hours, and the number of washing times is 3 - 5 times.

[0023] Preferably, after the separation in step 5), it further includes recovering the deep eutectic solvent; recovering the deep eutectic solvent is to collect the liquid after filtration separation, evaporate it, and collect the remaining components.

[0024] Preferably, the temperature of evaporation is 60 - 70°C.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0026] 1) The present invention can prepare aloe-emodin from the epidermis of aloe leaves in one step, with simple steps, short time, low energy consumption, high efficiency, and without the use of toxic organic solvents during the preparation process, without generating acid-base waste liquid, without using ferric chloride, without generating wastewater containing divalent iron, without pollution, and being green and environmentally friendly.

[0027] 2) Compared with the traditional method for preparing aloe-emodin, the present invention does not require the use of acid-base solutions and enzyme preparations during the preparation process, is environmentally friendly and has low costs. The preparation cost is low.

[0028] 3) The aloe-emodin prepared by the method of the present invention has high purity and strong free radical scavenging ability, and can be used as an antioxidant natural product.

[0029] 4) The deep eutectic solvent (Ch / La) of the present invention has a wide polarity, can well dissolve the cell wall and cell membrane of the aloe leaf epidermis, cause the depolymerization of the cell wall structure and the change of cell membrane permeability, and is more capable of extracting more aloin and aloe-emodin from the aloe leaf epidermis.

[0030] 5) The protons in the deep eutectic solvent (Ch / La) of the present invention contact with the aloin molecules, promoting the hydrolysis of the glycosyl part of the aloin molecules, thereby accelerating the conversion of aloin to aloe-emodin.

[0031] 6) The electroporation effect of the pulsed electric field of the present invention and the fact that protons will migrate faster around the aloin molecules under the action of the pulsed electric field strengthen the effect of the deep eutectic solvent, significantly reduce the time for preparing aloe-emodin by the present invention, and make the purity of aloe-emodin in the final product high, and the yield of aloe-emodin by the method of the present invention is high. Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the hydrolysis of aloin by pulsed electric field in cooperation with deep eutectic solvent (Ch / La). Detailed Embodiments

[0033] To better understand the present invention, the present invention will be further described below in conjunction with the drawings and embodiments, but the embodiments of the present invention are not limited thereto.

[0034] Figure 1 It is a schematic diagram of the mechanism of a method for extracting and preparing high-purity aloe-emodin from aloe skin in one step according to the present invention; as Figure 1 can be seen, the pulsed electric field in cooperation with the deep eutectic solvent (Ch / La) mainly acts on the C-C carbon-glycosidic bond of aloin to break it. After the C-C carbon-glycosidic bond of aloin is broken, the glucose group is removed to complete hydrolysis, and then it is oxidized to aloe-emodin.

[0035] Specifically, from Figure 1It can be seen that the deep eutectic solvent (Ch / La) formed by the hydrogen bond association of choline chloride and lactic acid in the method of the present invention has a wide polarity. It not only helps aloin and emodin to be better dispersed in the reaction system, increasing their solubility, but also can interact with the components of the aloe skin cell wall and cell membrane, making the cell wall structure loose and changing the permeability of the cell membrane. Combining with the electroporation effect of pulsed electric field, it can instantaneously destroy the plant cell membrane, making it easier for aloin and emodin to diffuse from the cell interior into the extraction solvent. Subsequently, the protons in the deep eutectic solvent (Ch / La) contact the aloin molecules, promoting the hydrolysis of the glycosyl part of the aloin molecules, thereby accelerating the conversion of the extracted aloin to emodin. Under the action of pulsed electric field, protons migrate faster around the aloin molecules, accelerating the hydrolysis reaction of the glycosyl in the aloin molecules, thus promoting the conversion of aloin to emodin. Based on the synergistic effect of pulsed electric field and deep eutectic solvent (Ch / La), the conversion of aloin in aloe skin to emodin is completed simultaneously during the treatment process, without the need for other treatment steps. In particular, it ensures both the yield and purity of emodin, and the yield of emodin reaches more than 4.9%, far higher than the prior art, proving excellent extraction effect. After the reaction, water is added to the deep eutectic solvent to destroy the stability of the deep eutectic solvent and decrease its solubility, so that the poorly water-soluble emodin can precipitate automatically, achieving the purpose of separation. After collecting and separating the precipitated emodin, the solution is rotary evaporated to separate the water, and then the deep eutectic solvent can be recycled, thus realizing no waste liquid generation in the whole reaction process.

[0036] In the prior art, there are also microwave treatment, ultrasonic extraction, etc. as strengthening measures for extracting emodin from aloe (Chinese Patent Application CN201710487520.3), but the results prove that these strengthening measures alone cannot simplify the extraction process, have high energy consumption, and the purity of the obtained extract is also low. Moreover, these measures do not involve the cooperation with deep eutectic reagents and do not reflect the synergistic effect of the above pulsed electric field and deep eutectic solvent.

[0037] Based on the above mechanism, a method for one-step extraction and preparation of high-purity emodin from aloe skin according to the present invention includes the following steps:

[0038] 1) Raw material preparation: Pretreat the aloe leaf epidermis;

[0039] 2) Preparation of deep eutectic solvent: Mix choline chloride and lactic acid, heat and stir until the solution is clear and transparent, and add pure water after cooling to obtain the deep eutectic solvent;

[0040] 3) Preparation of raw material solution: Mix the treated aloe leaf epidermis with the deep eutectic solvent to obtain the raw material solution;

[0041] 4) Pulsed electric field treatment: Place the raw material solution in a pulsed electric field device for treatment;

[0042] 5) Separation: Centrifuge the product obtained by pulsed electric field treatment, add water to the liquid phase after separation, refrigerate to obtain a precipitate, filter and separate the precipitate, wash it, and then freeze-dry it to obtain high-purity aloe-emodin.

[0043] As Figure 1 The mechanism reflects that the main technical measures in the above method are the synergistic cooperation of deep eutectic solvents and pulsed electric field treatment. As for the dosage ratio of choline chloride and lactic acid, the specific process conditions of the pulsed electric field can be obtained by combining the purpose of the present invention, brief experiments, and the conventional practice of pulsed electric fields; preferably, the molar ratio of choline chloride to lactic acid is (1:1)-(1:5); the treatment time of the pulsed electric field equipment is 10-20 min, the electric field frequency is 150-200 Hz, and the excitation voltage is 95-125 V.

[0044] The pretreatment in the method of the present invention is the basic practice of aloe leaf treatment. Preferably, the pretreatment is to wash the epidermis of aloe leaves, remove the surface dirt, and cut it into small pieces for standby; further preferably, the epidermis of aloe leaves is the epidermis of fresh Aloe arborescens leaves; the cut small pieces are 7×7 mm - 11×11 mm small pieces.

[0045] The purpose of heating and stirring in the method of the present invention is to make the solution clear and transparent. Through testing, the preferred heating temperature is 85-95 °C, the rotation speed is 200-400 rpm, and the heating and stirring time is 1-2 hours. A better practice is to stir in a water bath.

[0046] The concentration of the deep eutectic solvent in the method of the present invention and the deep eutectic solvent can be easily obtained by combining the purpose of the invention and testing. Preferably, the mass percentage concentration of the deep eutectic solvent is 70%-80%; in grams and milliliters respectively, preferably, the mass ratio of the aloe leaf epidermis to the volume of the deep eutectic solvent is (1:3)-(1:10)

[0047] As for the centrifugation process and refrigeration process in the method of the present invention, they can be obtained by combining the purpose of the invention and conventional processes. Preferably, the centrifugal force for centrifugal separation is 5000-10000 rpm, and the volume of water added is 4-8 times the volume of the liquid after centrifugal separation; the refrigeration temperature is 1-7 °C, and the refrigeration time is 1-5 hours.

[0048] One advantage of the present invention is that the deep eutectic solvent can be recycled, and the recycling method is also a relatively common practice, that is, collecting the liquid after filtration and separation, evaporating, and collecting the remaining components. Preferably, the evaporation temperature is 60-70 °C.

[0049] In the examples, the pulsed electric field equipment used is the SY-500 type pulsed electric field equipment of Guangzhou Paihu Technology Co., Ltd.

[0050] Example 1

[0051] A method for extracting and preparing high-purity aloe-emodin from aloe leaf epidermis in one step by using pulsed electric field combined with deep eutectic solvent, comprising the following steps:

[0052] Raw material preparation: The epidermis of fresh Aloe arborescens leaves is used as the raw material, washed to remove surface dirt, and cut into small pieces of 7×7 mm for standby;

[0053] Preparation of deep eutectic solvent: Choline chloride and lactic acid are mixed in a molar ratio of 1:1, and then placed in a water bath at 85 °C and stirred at a rotation speed of 200 rpm until a clear and transparent deep eutectic solvent is formed; then pure water is added to adjust the deep eutectic solvent to a mass percentage concentration of 70% for standby;

[0054] Sample preparation: Mix 10 g of aloe leaf epidermis blocks with 30 mL of deep eutectic solvent with a mass percentage concentration of 70% according to a solid-liquid ratio of 1:3 and set aside;

[0055] Pulsed electric field treatment: Put the 70% (g / g) deep eutectic solvent mixed with aloe leaf epidermis into a pulsed electric field device and treat it for 10 min, with an electric field frequency of 150 Hz and an excitation voltage of 95 V;

[0056] Separation of the sample: Use a centrifuge at 5000 rpm to separate the solid and liquid of the treated sample. Add 4 times the volume of ultrapure water to the separated liquid and refrigerate it at 1 °C for 1 hour; filter and separate the precipitate, wash it 3 times with ultrapure water and then freeze-dry to obtain aloe-emodin;

[0057] Recovery of deep eutectic solvent: Collect the liquid separated by filtration, pour it into a rotary evaporation device, and rotate and evaporate the water in the mixed liquid at a temperature of 60 °C; until all the water in the mixed liquid is evaporated (no longer dripping), after separation, the collected high-viscosity component is the successfully recovered deep eutectic solvent.

[0058] Comparative example 1

[0059] Raw material preparation: Select the epidermis of fresh Aloe arborescens leaves as the raw material, wash to remove surface dirt, and cut into small pieces of 7×7 mm for standby;

[0060] Preparation of aloin: Mix 10 g of aloe leaf epidermis blocks with 30 mL of hydrochloric acid solution with a mass percentage concentration of 6% according to a solid-liquid ratio of 1:3, heat at 98 °C for 20 min, cool to room temperature, filter, wash the filter residue 3 times with ultrapure water, combine the filtrate and washing liquid and add water to a constant volume of 1000 mL to obtain an aloin hydrochloride solution.

[0061] Preparation of aloe-emodin by hydrolysis oxidation reaction: Dissolve 75 g of ferric chloride in 120 mL of ultrapure water to obtain an aqueous solution of ferric chloride; heat the aloin hydrochloride solution to 98 °C, slowly add the prepared aqueous solution of ferric chloride, keep the temperature under reflux for 8 h. After the reaction, filter, wash the filter cake with ultrapure water three times until the filtrate has no obvious color, and combine the filtrates.

[0062] Purification: After drying the filter cake at 60 °C, add 400 mL of toluene, reflux and extract for 8 h. After the extraction, collect the toluene solution, filter it while it is hot with filter paper, and crystallize after the filtrate cools to obtain aloe-emodin.

[0063] Comparative Example 2

[0064] Raw material preparation: Select the epidermis of fresh Aloe arborescens leaves as raw materials, wash and remove the surface dirt, cut into small pieces of 7×7 mm for standby;

[0065] Enzymolysis: Add 2 times the mass of water to wet 10 g of aloe leaf epidermis blocks, and then add 0.5% of the mass of the aloe leaf epidermis blocks of cellulase and enzymolyze at 50 °C for 5 h to obtain an enzymolyzed material;

[0066] Extraction: Add 30 mL of an ethanol aqueous solution with a volume percentage concentration of 75% to the enzymolyzed material, and extract at 90 °C, and concentrate the extract to a specific gravity of 1.2 for the extract;

[0067] Alkaline alcohol dissolution: Add 50 times the mass of the extract to an ethanol aqueous solution with a volume percentage concentration of 90%, and then add 30% of the mass of the extract and a sodium carbonate solution with a mass concentration of 10% to dissolve for 5 h, the dissolution temperature is 90 °C, filter to obtain a filtrate;

[0068] Acid precipitation: Place the filtrate at room temperature, adjust the pH to 3 with hydrochloric acid with a volume percentage concentration of 22%, let it stand and precipitate for 2 h, filter to obtain the precipitate, and wash and dry the precipitate to obtain aloe-emodin.

[0069] Comparative Example 3

[0070] Raw material preparation: Use the epidermis of fresh Aloe arborescens leaves as raw materials, wash and remove the surface dirt, cut into small pieces of 7×7 mm for standby;

[0071] Sample preparation: Mix 10 g of aloe leaf epidermis blocks with 30 mL of an ethanol aqueous solution with a volume percentage concentration of 70% according to a material-liquid ratio of 1:3 for standby;

[0072] Pulsed electric field treatment: Put the 70% (v / v) ethanol aqueous solution mixed with aloe leaf epidermis into a pulsed electric field device and treat for 10 min, the electric field frequency is 150 Hz, and the excitation voltage is 95 V;

[0073] Separate the sample: Use a centrifuge at 5000 rpm to separate the solid and liquid of the treated sample;

[0074] Prepare crude aloe-emodin: Collect the liquid separated by filtration, pour it into a rotary evaporation device, and rotary evaporate the alcohol solvent in the mixed solution at 50 °C; until all the alcohol solvent in the mixed solution is evaporated to dryness (no longer dripping), after separation, a concentrate containing aloe-emodin is obtained, and then the concentrate is freeze-dried to obtain crude aloe-emodin;

[0075] Purification: Completely dissolve the crude aloe-emodin in 400 ml of toluene at 50 °C, then add an appropriate amount of activated carbon (10% of the mass of the crude product), and carry out decolorization treatment for 1 h. Filter while it is hot to remove the activated carbon and insoluble impurities, and the filtrate is cooled and crystallized, and aloe-emodin is obtained after drying.

[0076] Comparative Example 4:

[0077] Raw material preparation: Use the fresh epidermis of Aloe arborescens Mill. leaves as raw materials, wash and remove the dirt on the surface, cut into small pieces of 7×7 mm and set aside;

[0078] Preparation of deep eutectic solvent: Mix choline chloride and lactic acid in a molar ratio of 1:1, then place it in a water bath at 85 °C and stir at a speed of 200 rpm until a clear and transparent deep eutectic solvent is formed; then add pure water to adjust the deep eutectic solvent to a mass percentage concentration of 70% for standby;

[0079] Extraction: Mix 10 g of aloe leaf epidermis blocks with 30 mL of deep eutectic solvent with a mass percentage concentration of 70% according to a solid-liquid ratio of 1:3, and let it stand for 10 min;

[0080] Separate the sample: Use a centrifuge at 5000 rpm to separate the solid and liquid of the treated sample, add 4 times the volume of ultrapure water to the separated liquid, and refrigerate at 1 °C for 1 hour; filter the precipitate, wash it 3 times with ultrapure water, and then freeze-dry to obtain aloe-emodin;

[0081] Recover the deep eutectic solvent: Collect the liquid separated by filtration, pour it into a rotary evaporation device, and rotary evaporate the water in the mixed solution at 60 °C; until all the water in the mixed solution is evaporated to dryness (no longer dripping), after separation, the collected high-viscosity component is the successfully recovered deep eutectic solvent.

[0082] Comparative Example 5:

[0083] Raw material preparation: Use the fresh epidermis of Aloe arborescens Mill. leaves as raw materials, wash and remove the dirt on the surface, cut into small pieces of 7×7 mm and set aside;

[0084] Sample pretreatment: Weigh 10 g of aloe leaf epidermis blocks, acid-hydrolyze them with 25 mL of 1 mol / L CH3COOH for 30 min, then add 65 mL of 80% C2H5OH and let it stand for 30 min;

[0085] Microwave treatment: Microwave the above extract at a power of 400 W and control the microwave time until the temperature of the extract reaches 75 °C;

[0086] Ultrasonic extraction: Ultrasonically extract the extract after microwave treatment at a power of 100 W and an extraction temperature of 35 °C for 35 min, then centrifuge to obtain the supernatant;

[0087] Secondary ultrasonic extraction: Add 32.5 mL of 80% C2H5OH to the residue in the above step for secondary ultrasonic extraction, extract at 35 °C for 15 min with a power of 100 W, and then centrifuge to obtain the supernatant;

[0088] Preparation of aloe-emodin: Mix the supernatants obtained by centrifugation twice and pour them into a rotary evaporation device. Rotate and evaporate to separate the alcohol solvent in the mixed solution at 50 °C; until all the alcohol solvent in the mixed solution is evaporated (no longer dripping), after separation, obtain a concentrate containing aloe-emodin, and then freeze-dry the concentrate to obtain aloe-emodin.

[0089] The effects of aloe-emodin preparation in the six methods are characterized by the yield and purity of aloe-emodin.

[0090] Yield of aloe-emodin (%) = mass of obtained aloe-emodin / mass of dry matter of fresh aloe leaf epidermis raw material used × 100

[0091] The purity of aloe-emodin is detected by high performance liquid chromatography. Weigh 1 mg of the prepared aloe-emodin sample, dissolve it in 1 mL of chromatographic grade methanol, filter through a 0.45 μm nylon membrane and set aside. The detection equipment is Agilent 1260 Infinity high performance liquid chromatograph, the chromatographic column model is Eclipse Plus C18 (100 mm × 4.6 mm, 3.5 μm), and 0.2% acetic acid water (pump A) and acetonitrile (pump B) with a volume percentage concentration are used as the detection mobile phase. The detection program is: 0.00 - 10.00 min, pump B: volume percentage 5% - 30%, 10.00 - 12.00 min, pump B: volume percentage 30% - 95%, 12.00 - 18.00 min, pump B: volume percentage 95% - 95%, 18.00 - 19.00 min, pump B: volume percentage 95% - 5%, 19.00 - 25.00 min, pump B: volume percentage 5% - 5%. The flow rate is 0.8 mL / min, the detection wavelength is 430 nm, and the detection temperature is 25 °C.

[0092] Purity of aloe-emodin (%) = actual detected concentration of the sample by liquid chromatography / sample concentration (1 mg / mL) × 100

[0093] The yields and purities of aloe-emodin prepared in Example 1 and Comparative Examples 1, 2, 3, 4, and 5 are shown in Table 1. The yield of aloe-emodin prepared by pulsed electric field combined with deep eutectic solvent in Example 1 is 4.96%, which is significantly higher than 4.18%, 2.98%, 1.94%, 2.59%, and 2.96% of the preparation methods in Comparative Examples 1, 2, 3, 4, and 5. The purity of aloe-emodin prepared by pulsed electric field combined with deep eutectic solvent in Example 1 is 97.69%, which is also 3.51%, 2.43%, 2.79%, 0.68%, and 15% higher than those of the preparation methods in Comparative Examples 1, 2, 3, 4, and 5, respectively.

[0094] It should be emphasized that the whole process of extracting aloin and hydrolyzing aloin to prepare aloe-emodin by pulsed electric field combined with deep eutectic solvent in Example 1 only takes about 10 minutes, and it does not require as much time (more than 2 hours) as the traditional methods in Comparative Examples 1, 2, and 5 to complete multiple steps to obtain aloe-emodin.

[0095] Compared with Comparative Example 1, Example 1 does not require a large amount of hydrochloric acid solution, ferric chloride aqueous solution, and toluene during the whole reaction process, and will not produce a large amount of toxic waste liquid subsequently, resulting in less environmental pollution.

[0096] Compared with Comparative Example 2, Example 1 does not require the use of expensive enzymes during the preparation process, nor will it produce acid-base waste liquid.

[0097] Compared with Comparative Example 5, Example 1 will not produce acid-base waste liquid during the whole preparation process. Moreover, the electropermeabilization effect of pulsed electric field instantaneously destroys the plant cell membrane. Compared with the thermal effect of microwave and the cavitation effect of ultrasound, it causes less damage to active ingredients. At the same time, the instantaneous high voltage and low energy consumption characteristics of pulsed electric field are more energy-efficient than continuous microwave / ultrasound.

[0098] In particular, the pulsed electric field and deep eutectic solvent of the present invention cooperate with and promote each other, resulting in an improvement in both yield and purity compared to their individual effects (Comparative Examples 3 and 4), especially a particularly significant increase in yield.

[0099] Table 1. Yield and purity results of aloe-emodin prepared by different preparation methods

[0100]

[0101] Example 2

[0102] A method for directly extracting and preparing high-purity aloe-emodin from aloe leaf epidermis in one step by using pulsed electric field combined with deep eutectic solvent, comprising the following steps:

[0103] Raw material preparation: The epidermis of fresh Aloe arborescens Mill. leaves is used as the raw material, washed to remove surface dirt, and cut into small pieces of 9×9 mm for standby.

[0104] Preparation of deep eutectic solvent: Choline chloride and lactic acid are mixed in a molar ratio of 1:3, and then placed in a water bath at 90 °C and stirred at a rotation speed of 300 rpm until a clear and transparent deep eutectic solvent is formed; then an appropriate amount of pure water is added to adjust the deep eutectic solvent to a mass percentage concentration of 75% for standby.

[0105] Sample preparation: Mix 10 g of Aloe leaf epidermis blocks with 65 mL of deep eutectic solvent with a mass percentage concentration of 75% according to a solid-liquid ratio of 1:6.5 and set aside.

[0106] Pulsed electric field treatment: Put the 75% (g / g) deep eutectic solvent mixed with Aloe leaf epidermis into a pulsed electric field device and treat it for 15 min, with an electric field frequency of 175 Hz and an excitation voltage of 110 V.

[0107] Separation of samples: Use a centrifuge at 7500 rpm to separate the solid and liquid of the treated samples. Add 6 times the volume of ultrapure water to the separated liquid and refrigerate it at 4 °C for 3 hours; filter and separate the precipitate, wash it 4 times with ultrapure water, and then freeze-dry to obtain emodin.

[0108] Recovery of deep eutectic solvent: Collect the liquid separated by filtration, pour it into a rotary evaporation device, and rotate and evaporate the water in the mixed solution at 65 °C; until all the water in the mixed solution is evaporated (no longer dripping), after separation, the collected high-viscosity component is the successfully recovered deep eutectic solvent.

[0109] Comparative Example 6

[0110] Raw material preparation: Select the epidermis of fresh Aloe arborescens Mill. leaves as the raw material, wash to remove surface dirt, and cut into small pieces of 9×9 mm for standby.

[0111] Preparation of aloin: Mix 10 g of Aloe leaf epidermis blocks with 65 mL of hydrochloric acid solution with a mass percentage concentration of 6% according to a solid-liquid ratio of 1:6.5, heat at 98 °C for 20 min, cool to room temperature, filter, wash the filter residue 3 times with ultrapure water, combine the filtrate and washing liquid, and add water to make up to 1000 mL to obtain an aloin hydrochloric acid solution.

[0112] Preparation of emodin by hydrolysis and oxidation reaction: Dissolve 75 g of ferric chloride in an appropriate amount (120 mL) of ultrapure water to obtain an aqueous ferric chloride solution. Heat the aloin hydrochloric acid solution to 98 °C, slowly add the prepared aqueous ferric chloride solution dropwise, keep the temperature at reflux for 8 h, after the reaction is completed, filter, wash the filter cake 3 times with ultrapure water until the filtrate has no obvious color, and combine the filtrate.

[0113] Purification: After drying the filter cake at 60 °C, 400 mL of toluene was added, and the mixture was refluxed for 8 h. After the extraction was completed, the toluene solution was collected and filtered through filter paper while it was still hot. The filtrate was cooled and crystallized to obtain aloe-emodin.

[0114] Comparative Example 7

[0115] Raw material preparation: Fresh epidermis of Aloe arborescens Mill. leaves was selected as the raw material, washed to remove surface dirt, and cut into small pieces of 9×9 mm for standby.

[0116] Enzymolysis: 2 times the mass of the Aloe arborescens Mill. leaf epidermis blocks (10 g) was added for wetting, and then 0.5% of the mass of the Aloe arborescens Mill. leaf epidermis blocks of cellulase was added and enzymolyzed at 50 °C for 5 h to obtain an enzymolyzed material.

[0117] Extraction: 65 mL of an ethanol aqueous solution with a volume percentage concentration of 75% was added to the enzymolyzed material, and extraction was carried out at 90 °C. The extract was concentrated to a specific gravity of 1.2 for the extract.

[0118] Alkali-alcohol dissolution: 50 times the mass of the extract was added, and an ethanol aqueous solution with a volume percentage concentration of 90% was added. Then, 30% of the mass of the extract and a sodium carbonate solution with a mass concentration of 10% were added to dissolve for 5 h at a dissolution temperature of 90 °C, and then filtered to obtain a filtrate.

[0119] Acid precipitation: The filtrate was placed at room temperature, and hydrochloric acid with a volume percentage concentration of 22% was added to adjust the pH to 3, and then left to settle for 2 h. The precipitate was filtered, rinsed, and dried to obtain aloe-emodin.

[0120] Comparative Example 8

[0121] Raw material preparation: Fresh epidermis of Aloe arborescens Mill. leaves was used as the raw material, washed to remove surface dirt, and cut into small pieces of 9×9 mm for standby.

[0122] Sample preparation: The Aloe arborescens Mill. leaf epidermis blocks (10 g) and an ethanol aqueous solution with a volume percentage concentration of 75% (65 mL) were mixed at a solid-liquid ratio of 1:6.5 for standby.

[0123] Pulsed electric field treatment: The 75% (v / v) ethanol aqueous solution mixed with the Aloe arborescens Mill. leaf epidermis was put into a pulsed electric field device and treated for 15 min. The electric field frequency was 175 Hz, and the excitation voltage was 110 V.

[0124] Separation of the sample: The treated sample was separated into solid and liquid by a centrifuge at 7500 rpm.

[0125] Preparation of crude aloe-emodin: collecting the filtered and separated liquid, pouring it into a rotary evaporator, and separating the alcohol solvent in the mixed liquid by rotary evaporation at a temperature of 50° C.; after separation until the alcohol solvent in the mixed liquid is completely evaporated to dryness (no longer dripping out), a concentrate containing aloe-emodin is obtained, and then the concentrate is freeze-dried to obtain the crude aloe-emodin;

[0126] Purification: After the crude aloe-emodin is completely dissolved in 400 ml of toluene at 50°C, an appropriate amount of activated carbon (10% of the crude quality) is added for decolorization for 1 hour. The activated carbon and insoluble impurities are removed by hot filtration, and the filtrate is cooled and crystallized, and then dried to obtain aloe-emodin.

[0127] Comparative Example 9

[0128] Raw material preparation: Use the fresh leaf skin of Aloe arborescens as raw material, wash and remove the dirt on the surface, cut into 9×9mm small pieces for later use;

[0129] Preparation of a low eutectic solvent: Choline chloride and lactic acid are mixed in a molar ratio of 1:3, and then stirred in a water bath at 90°C and 300 rpm until a clear and transparent low eutectic solvent is formed; then, an appropriate amount of pure water is added to adjust the low eutectic solvent to a mass percentage concentration of 75% for later use;

[0130] Extraction: Aloe leaf epidermis (10 g) was mixed with a 75% eutectic solvent (65 mL) at a solid-liquid ratio of 1:6.5 and allowed to stand for 15 min.

[0131] Separate the sample: Use a centrifuge at 7500rpm to separate the solid and liquid of the treated sample, add 6 times the volume of ultrapure water to the separated liquid, and refrigerate at 4°C for 3 hours; filter and separate the precipitate, wash it 4 times with ultrapure water, and then freeze-dry it to obtain aloe-emodin;

[0132] Recover the low eutectic solvent: collect the filtered and separated liquid, pour it into a rotary evaporator, and separate the water in the mixed liquid by rotary evaporation at 65°C; after separation, the high-viscosity component collected is the successfully recovered low eutectic solvent.

[0133] Comparative Example 10

[0134] Raw material preparation: Use the fresh leaf skin of Aloe arborescens as raw material, wash and remove the dirt on the surface, cut into 7×7mm small pieces for later use;

[0135] Sample pretreatment: weigh 10g of aloe leaf epidermis, acid hydrolyze with 25mL 1mol / L CH3COOH for 30min, then add 65mL 80% C2H5OH and let stand for 30min;

[0136] Microwave treatment: The above extract was treated with microwave at a power of 400 W, and the microwave time was controlled until the temperature of the extract reached 75 °C;

[0137] Ultrasonic extraction: The extract after microwave treatment was extracted under the conditions of ultrasonic power of 100 W and extraction temperature of 40 °C for 35 min, and then centrifuged to obtain the supernatant;

[0138] Secondary ultrasonic extraction: 32.5 mL of 80% C2H5OH was added to the residue in the above step for secondary ultrasonic extraction. It was extracted at 40 °C for 15 min with an ultrasonic power of 100 W, and then centrifuged to obtain the supernatant;

[0139] Preparation of aloe-emodin: The supernatants obtained by centrifugation twice were mixed and poured into a rotary evaporation device. The alcohol solvent in the mixed solution was separated by rotary evaporation at 50 °C; until all the alcohol solvent in the mixed solution was evaporated to dryness (no longer dripping), after separation, a concentrate containing aloe-emodin was obtained, and then the concentrate was freeze-dried to obtain aloe-emodin.

[0140] The effects of aloe-emodin preparation in the six methods were characterized by the yield and purity of aloe-emodin.

[0141] Yield of aloe-emodin (%) = mass of obtained aloe-emodin / mass of dry matter of fresh aloe leaf epidermis raw material used × 100

[0142] The purity of aloe-emodin was detected by high performance liquid chromatography. 1 mg of the prepared aloe-emodin sample was weighed and dissolved in 1 mL of chromatographic grade methanol, and then passed through a 0.45 μm nylon membrane for standby. The detection equipment was an Agilent 1260 Infinity high performance liquid chromatograph, and the chromatographic column model was Eclipse Plus C18 (100 mm × 4.6 mm, 3.5 μm). The mobile phase for detection was 0.2% acetic acid water (pump A) and acetonitrile (pump B) with a volume percentage concentration. The detection procedure was as follows: 0.00 - 10.00 min, pump B: volume percentage 5% - 30%, 10.00 - 12.00 min, pump B: volume percentage 30% - 95%, 12.00 - 18.00 min, pump B: volume percentage 95% - 95%, 18.00 - 19.00 min, pump B: volume percentage 95% - 5%, 19.00 - 25.00 min, pump B: volume percentage 5% - 5%. The flow rate was 0.8 mL / min, the detection wavelength was 430 nm, and the detection temperature was 25 °C.

[0143] Purity of aloe-emodin (%) = actual detected concentration of the sample by liquid chromatography / sample concentration (1 mg / mL) × 100

[0144] The yields and purities of aloe-emodin prepared in Example 2 and Comparative Examples 6, 7, 8, 9, and 10 are shown in Table 2. The yield of aloe-emodin prepared by combining pulsed electric field with deep eutectic solvent in Example 2 was 5.09%, higher than 4.46%, 3.27%, 2.07%, 2.86%, and 3.06% of the preparation methods in Comparative Examples 6, 7, 8, 9, and 10. The purity of aloe-emodin prepared by combining pulsed electric field with deep eutectic solvent in Example 2 was 98.29%, also 2.96%, 2.14%, 2.42%, 0.79%, and 15.54% higher than those of the preparation methods in Comparative Examples 6, 7, 8, 9, and 10, respectively.

[0145] The entire process of extracting aloin and hydrolyzing aloin to prepare aloe-emodin by combining pulsed electric field with deep eutectic solvent in Example 2 only takes about 15 minutes, and does not require as much time (more than 2 hours) as the traditional methods in Comparative Examples 6, 7, and 10 to complete multiple steps to obtain aloe-emodin. And compared with Comparative Example 6, Example 2 does not require a large amount of hydrochloric acid solution, ferric chloride aqueous solution, and toluene during the whole reaction process, and will not produce a large amount of toxic waste liquid afterwards, causing little pollution to the environment. Compared with Comparative Example 7, Example 2 does not require the use of expensive enzymes during the preparation process, nor will it produce acid-base waste liquid. Compared with Comparative Example 10, Example 2 will not produce acid-base waste liquid during the whole preparation process. Moreover, the electropermeation effect of pulsed electric field instantaneously destroys the plant cell membrane. Compared with the thermal effect of microwave and the cavitation effect of ultrasound, it causes less damage to the active ingredients. At the same time, the characteristic of high voltage and low energy consumption of pulsed electric field is more energy-saving than continuous microwave / ultrasound. In addition, pulsed electric field and deep eutectic solvent cooperate with each other and promote each other, producing a more significant effect than their individual actions (Comparative Examples 8 and 9).

[0146] Table 2. Results of yields and purities of aloe-emodin prepared by different preparation methods

[0147]

[0148] Example 3

[0149] A method for directly extracting and preparing high-purity aloe-emodin from the epidermis of aloe leaves in one step by combining pulsed electric field with deep eutectic solvent, comprising the following steps:

[0150] Raw material preparation: The epidermis of fresh Aloe arborescens Mill. leaves is used as the raw material, washed to remove surface dirt, and cut into small pieces of 11×11 mm for standby;

[0151] Preparation of deep eutectic solvent: Choline chloride and lactic acid are mixed in a molar ratio of 1:5, and then placed in a water bath at 95°C and stirred at a rotation speed of 400 rpm until a clear and transparent deep eutectic solvent is formed; then an appropriate amount of pure water is added to adjust the deep eutectic solvent to a mass percentage concentration of 80% for standby;

[0152] Sample preparation: Mix 10 g of aloe leaf epidermis blocks with 100 mL of eutectic solvent with a mass percentage concentration of 80% at a solid-liquid ratio of 1:10, and set aside for later use.

[0153] Pulsed electric field treatment: Put the 80% (g / g) eutectic solvent mixed with aloe leaf epidermis into a pulsed electric field device for 20 min, with an electric field frequency of 200 Hz and an excitation voltage of 125 V.

[0154] Separation of the sample: Use a centrifuge at 10000 rpm to separate the solid and liquid of the treated sample. Add 8 times the volume of ultrapure water to the separated liquid and refrigerate it at 7 °C for 5 hours. Filter and separate the precipitate, wash it 5 times with ultrapure water, and then freeze-dry to obtain emodin.

[0155] Recovery of eutectic solvent: Collect the liquid separated by filtration, pour it into a rotary evaporation device, and rotate and evaporate the water in the mixed solution at 70 °C until all the water in the mixed solution is evaporated (no longer dripping). After separation, the collected high-viscosity component is the successfully recovered eutectic solvent.

[0156] Comparative Example 11

[0157] Raw material preparation: Select the epidermis of fresh Aloe arborescens leaves as the raw material, wash and remove the surface dirt, and cut it into small pieces of 11×11 mm for later use.

[0158] Preparation of aloin: Mix 10 g of aloe leaf epidermis blocks with 100 mL of hydrochloric acid solution with a mass percentage concentration of 6% at a solid-liquid ratio of 1:10, heat at 98 °C for 20 min, cool to room temperature, filter, wash the filter residue 3 times with ultrapure water, combine the filtrate and washing liquid, and add water to make up to 1000 mL to obtain aloin hydrochloride solution.

[0159] Preparation of emodin by hydrolysis and oxidation reaction: Dissolve 75 g of ferric chloride in an appropriate amount (120 mL) of ultrapure water to obtain an aqueous ferric chloride solution. Heat the aloin hydrochloride solution to 98 °C, slowly add the prepared aqueous ferric chloride solution dropwise, keep it under reflux for 8 h. After the reaction, filter, wash the filter cake 3 times with ultrapure water until the filtrate has no obvious color, and combine the filtrate.

[0160] Purification: After drying the filter cake at 60 °C, add 400 mL of toluene, reflux and extract for 8 h. After the extraction, collect the toluene solution, filter it while it is hot with filter paper, and crystallize the filtrate after cooling to obtain emodin.

[0161] Comparative Example 12

[0162] Raw material preparation: Select the epidermis of fresh Aloe arborescens leaves as the raw material, wash and remove the surface dirt, and cut it into small pieces of 11×11 mm for later use.

[0163] Enzymatic hydrolysis: Add twice the mass of wetting agent to the aloe leaf epidermis blocks (10 g), and then add 0.5% of the mass of the aloe leaf epidermis blocks of cellulase and perform enzymatic hydrolysis at 50 °C for 5 h to obtain an enzymatically hydrolyzed material;

[0164] Extraction: Add 100 mL of an ethanol aqueous solution with a volume percentage concentration of 75% to the enzymatically hydrolyzed material, perform extraction at 90 °C, and concentrate the extract to a specific gravity of 1.2 for the extract;

[0165] Alkali-alcohol dissolution: Add 50 times the mass of the extract, and an ethanol aqueous solution with a volume percentage concentration of 90%, then add 30% of the mass of the extract and a sodium carbonate solution with a mass concentration of 10% to dissolve for 5 h, with the dissolution temperature at 90 °C, filter to obtain a filtrate;

[0166] Acid precipitation: Let the filtrate stand at room temperature, adjust the pH to 3 with hydrochloric acid with a volume percentage concentration of 22%, let it stand for precipitation for 2 h, filter to obtain the precipitate, and after rinsing and drying the precipitate, aloe-emodin is obtained.

[0167] Comparative Example 13

[0168] Raw material preparation: The epidermis of fresh Aloe arborescens Mill. leaves is used as the raw material, washed to remove surface dirt, and cut into small pieces of 11×11 mm for standby;

[0169] Sample preparation: Mix 10 g of aloe leaf epidermis blocks with 100 mL of an ethanol aqueous solution with a volume percentage concentration of 80% according to a material-liquid ratio of 1:10 and set aside;

[0170] Pulsed electric field treatment: Put the 80% (v / v) ethanol aqueous solution mixed with aloe leaf epidermis into a pulsed electric field device and treat for 20 min, with the electric field frequency of 200 Hz and the excitation voltage of 125 V;

[0171] Separate the sample: Use a centrifuge at 10000 rpm to separate the solid and liquid of the treated sample;

[0172] Prepare the crude product of aloe-emodin: Collect the filtered and separated liquid, pour it into a rotary evaporation device, and rotate and evaporate the alcohol solvent in the mixed solution at 50 °C; until all the alcohol solvent in the mixed solution is evaporated (no longer dripping) after separation, a concentrate containing aloe-emodin is obtained, and then the concentrate is freeze-dried to obtain the crude product of aloe-emodin;

[0173] Purification: Completely dissolve the crude product of aloe-emodin in 400 ml of toluene at 50 °C, then add an appropriate amount of activated carbon (10% of the mass of the crude product), and perform decolorization treatment for 1 h. Filter while it is hot to remove the activated carbon and insoluble impurities, cool the filtrate to crystallize, and obtain aloe-emodin after drying.

[0174] Comparative Example 14

[0175] Raw material preparation: The epidermis of fresh Aloe arborescens Mill. leaves is used as the raw material. Wash it to remove the surface dirt and cut it into small pieces of 11×11 mm for standby;

[0176] Preparation of deep eutectic solvent: Choline chloride and lactic acid are mixed in a molar ratio of 1:5, and then placed in a water bath at 95 °C with a rotation speed of 400 rpm and stirred until a clear and transparent deep eutectic solvent is formed; then an appropriate amount of pure water is added to adjust the deep eutectic solvent to a mass percentage concentration of 80% for standby;

[0177] Extraction: Mix 10 g of aloe leaf epidermis blocks with 100 mL of deep eutectic solvent with a mass percentage concentration of 80% according to a solid-liquid ratio of 1:10, and then let it stand for 20 min;

[0178] Separation of sample: Use a centrifuge at 10000 rpm to separate the solid and liquid of the treated sample. Add 8 times the volume of ultrapure water to the separated liquid and refrigerate it at 7 °C for 5 hours; filter and separate the precipitate, wash it 5 times with ultrapure water, and then freeze-dry to obtain emodin;

[0179] Recovery of deep eutectic solvent: Collect the liquid after filtration and separation, pour it into a rotary evaporation device, and rotate and evaporate the water in the mixed solution at 70 °C; until all the water in the mixed solution is evaporated (no longer dripping), the collected high-viscosity component is the successfully recovered deep eutectic solvent.

[0180] Control Example 15

[0181] Raw material preparation: The epidermis of fresh Aloe arborescens Mill. leaves is used as the raw material. Wash it to remove the surface dirt and cut it into small pieces of 7×7 mm for standby;

[0182] Sample pretreatment: Weigh 10 g of aloe leaf epidermis blocks, acidify them with 25 mL of 1 mol / L CH3COOH for 30 min, and then add 65 mL of 80% C2H5OH and let it stand for 30 min;

[0183] Microwave treatment: The microwave treatment power of the above extraction solution is 400 W, and the microwave time is controlled until the temperature of the extraction solution reaches 75 °C;

[0184] Ultrasonic extraction: The extraction solution after microwave treatment is extracted at an ultrasonic power of 100 W and an extraction temperature of 45 °C for 35 min, and then centrifuged to obtain the supernatant;

[0185] Second ultrasonic extraction: Add 32.5 mL of 80% C2H5OH to the residue in the above step for second ultrasonic extraction, extract at 45 °C for 15 min, with an ultrasonic power of 100 W, and then centrifuge to obtain the supernatant;

[0186] Preparation of aloe-emodin: The supernatants obtained from the two centrifugations were mixed and then poured into a rotary evaporation device. The alcohol solvent in the mixed solution was separated by rotary evaporation at 50 °C until all the alcohol solvent in the mixed solution was evaporated to dryness (no longer dripping). After separation, a concentrate containing aloe-emodin was obtained, and then the concentrate was freeze-dried to obtain aloe-emodin.

[0187] The preparation effects of aloe-emodin in the six methods were characterized by the yield and purity of aloe-emodin.

[0188] Yield of aloe-emodin (%) = mass of obtained aloe-emodin / mass of dry matter of fresh aloe leaf epidermis raw material used × 100

[0189] The purity of aloe-emodin was detected by high performance liquid chromatography. Weigh 1 mg of the prepared aloe-emodin sample and dissolve it in 1 mL of chromatographic grade methanol. After passing through a 0.45 μm nylon membrane, it was reserved for use. The detection equipment was an Agilent 1260 Infinity high performance liquid chromatograph, and the chromatographic column model was Eclipse Plus C18 (100 mm × 4.6 mm, 3.5 μm). Aqueous acetic acid with a volume percentage concentration of 0.2% (pump A) and acetonitrile (pump B) were used as the detection mobile phase. The detection procedure was as follows: 0.00 - 10.00 min, pump B: volume percentage 5% - 30%; 10.00 - 12.00 min, pump B: volume percentage 30% - 95%; 12.00 - 18.00 min, pump B: volume percentage 95% - 95%; 18.00 - 19.00 min, pump B: volume percentage 95% - 5%; 19.00 - 25.00 min, pump B: volume percentage 5% - 5%. The flow rate was 0.8 mL / min, the detection wavelength was 430 nm, and the detection temperature was 25 °C.

[0190] Purity of aloe-emodin (%) = actual detected concentration of the sample by liquid chromatography / sample concentration (1 mg / mL) × 100

[0191] The results of the yield and purity of aloe-emodin prepared in Example 3 and Comparative Examples 11, 12, 13, 14, and 15 are shown in Table 3. The yield of aloe-emodin prepared by pulsed electric field combined with deep eutectic solvent in Example 3 was 5.48%, which was higher than 4.34%, 3.19%, 2.24%, 3.16%, and 3.09% of the preparation methods in Comparative Examples 11, 12, 13, 14, and 15.

[0192] The purity of aloe-emodin prepared by pulsed electric field combined with deep eutectic solvent in Example 3 was 98.44%, which was also 3.39%, 2.38%, 2.52%, 0.41%, and 15.55% higher than the preparation methods in Comparative Examples 11, 12, 13, 14, and 15, respectively.

[0193] Table 3. Yield and purity results of aloe-emodin prepared by different preparation methods

[0194]

[0195] In Example 3, the entire process of extracting aloin to hydrolyzing aloin to prepare aloe-emodin by pulsed electric field combined with deep eutectic solvent only takes about 20 minutes, without the need for a long time (more than 2 hours) as in the traditional methods of Comparative Examples 11, 12, and 15 to complete multiple steps to obtain aloe-emodin. Moreover, compared with Comparative Example 11, Example 3 does not require a large amount of hydrochloric acid solution, ferric chloride aqueous solution, and toluene during the entire reaction process, and will not produce a large amount of toxic waste liquid subsequently, resulting in less environmental pollution. Compared with Comparative Example 12, Example 3 does not need to use expensive enzymes during the preparation process and will not produce acid-base waste liquid. Compared with Comparative Example 15, Example 3 not only does not produce acid-base waste liquid during the entire preparation process, but also the electro-poration effect of pulsed electric field instantaneously destroys the plant cell membrane. Compared with the thermal effect of microwave and the cavitation effect of ultrasound, it causes less damage to the active ingredients. At the same time, the instantaneous high voltage and low energy consumption characteristics of pulsed electric field are more energy-efficient than continuous microwave / ultrasound.

Claims

1. A method for one-step extraction and preparation of high-purity aloe-emodin from aloe skin, characterized in that It includes the following steps: 1) Raw material preparation: Pretreat the aloe leaf epidermis; 2) Preparation of deep eutectic solvent: Mix choline chloride and lactic acid, heat and stir until the solution is clear and transparent, and add pure water after cooling to obtain the deep eutectic solvent; 3) Preparation of raw material solution: Mix the pretreated aloe leaf epidermis with the deep eutectic solvent to obtain the raw material solution; 4) Pulsed electric field treatment: Place the raw material solution in a pulsed electric field device for treatment; 5) Separation: Centrifuge the product obtained by pulsed electric field treatment, add water to the separated liquid phase, refrigerate to obtain a precipitate, filter and separate the precipitate, wash it, and then freeze-dry it to obtain high-purity aloe-emodin.

2. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, wherein, In step 1), the pretreatment is to wash the aloe leaf epidermis, remove the surface dirt, and cut it into small pieces for later use.

3. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 2, characterized in that, The aloe leaf epidermis is the epidermis of fresh Aloe arborescens Mill. leaves; the cut small pieces are 7×7mm - 11×11mm small pieces.

4. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, characterized in that, In step 2), the molar ratio of choline chloride to lactic acid is (1:1) - (1:5); The heating and stirring are carried out in a water bath at a temperature of 85 - 95°C and a rotation speed of 200 - 400 rpm; the heating and stirring time is 1 - 2 hours.

5. A method for extracting and preparing high-purity aloe-emodin from aloe leaf epidermis in one step according to claim 1, characterized in that, In step 2), the mass percentage concentration of the deep eutectic solvent is 70% - 80%.

6. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, wherein In step 3), taking grams and milliliters as units respectively, the mass ratio of the aloe leaf epidermis to the volume of the deep eutectic solvent is (1:3) - (1:10).

7. A method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, characterized in that, In step 4), the treatment time of the pulsed electric field device is 10 - 20 min, the electric field frequency is 150 - 200 Hz, and the excitation voltage is 95 - 125 V.

8. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, wherein In step 5), the centrifugal force for centrifugal separation is 5000 - 10000 rpm, and the volume of water added is 4 - 8 times the volume of the liquid after centrifugal separation; The refrigeration temperature is 1 - 7°C, the refrigeration time is 1 - 5 hours, and the number of washing times is 3 - 5 times.

9. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 1, characterized in that, After separation in step 5), it also includes recovering the deep eutectic solvent; recovering the deep eutectic solvent is to collect the liquid after filtration separation, evaporate it, and collect the remaining components.

10. The method for one-step extraction and preparation of high-purity aloe-emodin from aloe leaf epidermis according to claim 9, characterized in that, The evaporation temperature is 60 - 70°C.

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