Method for separating flavone and saponin from wild jujube leaves and application of flavone and saponin

Abstract

The invention belongs to the technical field of separation and extraction, and particularly relates to a method for separating flavone and saponin from wild jujube leaves as well as a preparation method and application of the flavone and the saponin. The method provided by the invention comprises the following steps: 1) mixing wild jujube leaf powder with water, carrying out first ultrasonic treatment, then carrying out first heat reflux extraction, and separating to obtain an extracting solution A and solid residues; 2) mixing the solid residues with an alcohol-water solution, carrying out second ultrasonic treatment, then carrying out second heat reflux extraction, and separating to obtain an extracting solution B; and (3) mixing the extracting solution A and the extracting solution B, concentrating to remove ethanol to form a concentrated solution, and then carrying out gradient elution on the concentrated solution by using a macroporous resin column formed by compounding D101 macroporous resin and HPD-600 macroporous resin to obtain the flavone-rich extract and the saponin-rich extract. According to the method, the flavone-rich extract and the saponin-rich extract can be simultaneously and efficiently extracted and separated from the wild jujube leaves.

Description

Technical Field

[0001] This invention belongs to the field of separation and extraction technology, specifically relating to a method for separating flavonoids and saponins from jujube leaves, its preparation method, and its application. Background Technology

[0002] Ziziphus jujuba var. spinosa is a traditional medicinal plant. Its seeds (ziziphus jujuba kernels) have the effects of nourishing the heart and calming the mind, and improving sleep. It is a traditional Chinese medicine. In recent years, studies have found that the buds and leaves of ziziphus jujuba are also rich in flavonoids (such as quercetin-3-O-rutin glycoside and kaempferol glycoside), saponins (such as ziziphus jujuba kernel saponins A and B) and iridoids, which have significant sedative and hypnotic effects. Therefore, ziziphus jujuba leaves are also known as "Oriental Sleeping Leaves". They have unique effects in calming the mind and improving sleep quality. However, in the current industrial product development, everyone only focuses on ziziphus jujuba kernels and ignores ziziphus jujuba leaves, which are rich in resources, low in cost and have high development value. The reason is that the extraction and purification of active ingredients from ziziphus jujuba buds and leaves still face the following challenges: (1) Complex composition: In addition to the target components, it also contains a large number of polysaccharides, proteins, pigments and other impurities, which affect the subsequent purification efficiency. (2) Large polarity difference: Flavonoids (medium polarity) and saponins (strong polarity) have different solubilities, and traditional single solvent extraction is difficult to achieve both. (3) Heat-sensitive components are easily degraded: Long-term high-temperature extraction may cause some active ingredients (such as certain saponins) to decompose, reducing the yield.

[0003] Currently, the main extraction methods for jujube buds and leaves include: (1) Traditional water extraction, which has the advantage of low cost and suitability for industrial production. However, the extraction efficiency is low, there are many water-soluble impurities such as polysaccharides, and purification is difficult. The water solubility of flavonoids is poor, resulting in insufficient recovery rate (usually <50%). (2) Single alcohol extraction, which has the advantage of good extraction effect on flavonoids. However, high concentrations of ethanol (such as above 70%) will inhibit the dissolution of saponins, resulting in incomplete extraction. (3) Supercritical CO2 extraction, which has the advantage of no solvent residue and suitability for heat-sensitive components. However, the equipment cost is high, limiting large-scale production. (4) Enzyme-assisted extraction, which has the advantage of degrading cell walls and improving extraction efficiency. However, the enzymatic hydrolysis conditions (pH, temperature) are strict, and the process stability is poor. Furthermore, existing purification technologies for jujube leaf extracts have shortcomings. Currently, the purification of jujube buds and leaves mainly relies on macroporous adsorption resins, but different resins have different adsorption effects. For example, AB-8 resin has a strong adsorption effect on saponins but a poor elution effect on flavonoids, resulting in low recovery rates; polyamide resins are suitable for flavonoid purification, but saponin loss occurs due to hydrogen bond selectivity. Therefore, existing jujube leaf extraction, separation, and purification technologies cannot simultaneously and efficiently enrich flavonoids and saponins. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect that the extraction, separation and purification technology of jujube leaves is difficult to simultaneously and efficiently enrich flavonoids and saponins, thereby providing a method for separating flavonoids and saponins from jujube leaves, as well as its preparation method and application.

[0005] This invention provides a method for separating flavonoids and saponins from jujube leaves, comprising the following steps: 1) Mix jujube leaf powder with water, perform a first ultrasonic treatment, and then perform a first hot reflux extraction to separate extract A and solid residue; 2) Mix the solid residue obtained in step 1) with the alcohol-water solution, subject it to a second ultrasonic treatment, and then perform a second hot reflux extraction to obtain extract B; 3) Mix the extract A obtained in step 1) and the extract B obtained in step 2), concentrate to remove ethanol to form a concentrate, and then use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin to perform gradient elution of the concentrate to obtain the flavonoid-rich extract and saponin-rich extract.

[0006] Preferably, in step 3), the mass ratio of D101 macroporous resin to HPD-600 macroporous resin in the macroporous resin column formed by the composite of D101 macroporous resin and HPD-600 macroporous resin is 1:(0.5-1.5). And / or, the gradient elution concentrate step includes: loading the concentrate into a macroporous resin column, and then eluting with water, an ethanol aqueous solution with a mass concentration of 8-12%, an ethanol aqueous solution with a mass concentration of 28-32%, an ethanol aqueous solution with a mass concentration of 48-52%, an ethanol aqueous solution with a mass concentration of 68-72%, and an ethanol aqueous solution with a mass concentration of 78-82%. After the gradient elution step is completed, the eluent with an ethanol aqueous solution of 28-32% by mass is concentrated to obtain a flavonoid-rich extract; the eluent with an ethanol aqueous solution of 48-52% by mass is concentrated to obtain a saponin-rich extract.

[0007] Preferably, the elution flow rate of the water elution is 1-3 BV / h, and the elution volume is 1-3 column volumes; The elution flow rate of the ethanol aqueous solution with a mass concentration of 8-12% is 1-3 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate for the ethanol aqueous solution with a mass concentration of 28-32% is 1.2-4 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate of the ethanol aqueous solution with a mass concentration of 48-52% is 2-4 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate of the ethanol aqueous solution with an ethanol mass concentration of 68-72% is 1.5-2.5 BV / h, and the elution volume is 1-3 column volumes. The elution flow rate of the ethanol aqueous solution with a mass concentration of 78-82% is 0.5-1.5 BV / h, and the elution volume is 1-2 column volumes.

[0008] Preferably, in step 3), the macroporous resin column formed by the composite of D101 macroporous resin and HPD-600 macroporous resin is soaked in alcohol solution and washed until there is no alcohol odor, soaked in alkaline solution and washed until neutral, and soaked in acid solution and washed until neutral. Preferably, the alcohol concentration in the alcohol solution is 90-99% by mass; The alcohol solution is selected from aqueous ethanol solution; And / or, the mass concentration of alkali in the alkaline solution is 3-8%; The alkaline solution is selected from an aqueous sodium hydroxide solution; And / or, the mass concentration of acid in the acid solution is 3-8%; The acid solution is selected from hydrochloric acid aqueous solution and / or phosphoric acid aqueous solution; And / or, the soaking time in the alcohol solution is 24-72 hours; And / or, the immersion time in the alkaline solution is 12-36 hours; And / or, the acid solution soaking time is 12-36 hours.

[0009] Preferably, the jujube leaf powder mentioned in step 1) is jujube leaf powder that has passed through a 20-40 mesh sieve; The ratio of jujube leaf powder to water in step 1) is 1:(18-22), with units of g / mL; The ultrasonic power of the first ultrasonic treatment is 300-600W, the ultrasonic treatment temperature is 48-52℃, and the ultrasonic treatment time is 0.5-2h. The first hot reflux extraction temperature is 70-90℃, and the first hot reflux extraction time is 1.5-2.5h.

[0010] Preferably, the ratio of the solid residue to the ethanol-water solution in step 2) is (0.5-1.1):(18-22), in g / mL; The alcohol-water solution has an alcohol concentration of 48-52% by mass. The aqueous alcohol solution is selected from aqueous ethanol solution; The ultrasonic power of the second ultrasonic treatment is 300-600W, the ultrasonic treatment temperature is 48-52℃, and the ultrasonic treatment time is 0.5-2h. The second hot reflux extraction temperature is 70-90℃, and the second hot reflux extraction time is 1.5-2.5h.

[0011] The concentration described in step 3) is vacuum concentration, which can be selected, and the concentration temperature is 50-80℃.

[0012] This invention provides a flavonoid-rich extract, obtained by the method described above for separating flavonoids and saponins from jujube leaves.

[0013] This invention provides a saponin-rich extract, obtained by the method described above for separating flavonoids and saponins from jujube leaves.

[0014] This invention provides a method for separating flavonoids and saponins from jujube leaves using the above-described flavonoid-rich extract and / or the above-described saponin-rich extract, and its application in sleep aid drug formulations.

[0015] The technical solution of this invention has the following advantages: The present invention provides a method for separating flavonoids and saponins from jujube leaves, comprising the following steps: 1) mixing jujube leaf powder with water, subjecting it to a first ultrasonic treatment, and then performing a first hot reflux extraction to separate extract A and solid residue; 2) mixing the solid residue obtained in step 1) with an alcohol-water solution, subjecting it to a second ultrasonic treatment, and then performing a second hot reflux extraction to separate extract B; 3) mixing extract A obtained in step 1) and extract B obtained in step 2), concentrating to remove ethanol to form a concentrate, and then using a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin to perform gradient elution of the concentrate to obtain the flavonoid-rich extract and saponin-rich extract. This invention employs ultrasound-assisted dual-solvent (water / alcohol-water solution) stepwise extraction: combining water extraction (enriching saponins) and alcohol extraction (enriching flavonoids), it takes into account components of different polarities. The ultrasound-assisted extraction utilizes the cavitation effect generated by the ultrasound waves to disrupt plant cell walls, making flavonoids (moderately polar) and saponins (strongly polar) more easily soluble. Simultaneously, the dual-solvent synergistic extraction prioritizes the dissolution of saponins and some polysaccharides in the water extraction stage (avoiding saponin precipitation caused by high concentrations of ethanol), while the alcohol-water extraction stage optimizes flavonoid dissolution and avoids high alcohol concentrations inhibiting saponin extraction. Furthermore, a gradient elution is achieved using a composite macroporous resin formed by mixing D101 macroporous resin and HPD-600 macroporous resin, precisely separating the flavonoid-rich extract and the saponin-rich extract. HPD-600 macroporous resin captures medium-sized components, such as flavonoids, while D101 macroporous resin adsorbs saponins. The synergistic effect of D101 and HPD-600 macroporous resins mitigates the loss of target components. Meanwhile, the pore sizes of the two macroporous resins are matched: the pore size of D101 (8-10nm) is suitable for the adsorption of saponins (molecular weight 800-1200Da), and the pore size of HPD-600 (9-12nm) is suitable for the adsorption of flavonoids (molecular weight 300-600Da); the method of the present invention can simultaneously and efficiently extract and separate flavonoid-rich extracts and saponin-rich extracts from jujube leaves.

[0016] The method for separating flavonoids and saponins from jujube leaves provided by this invention, in step 3), the mass ratio of D101 macroporous resin to HPD-600 macroporous resin in the macroporous resin column formed by the composite of D101 macroporous resin and HPD-600 macroporous resin is 1:(0.5-1.5); and / or, the gradient elution concentration step includes: loading the concentrate into the macroporous resin column, and then eluting with water, ethanol aqueous solution with a mass concentration of 8-12%, ethanol aqueous solution with a mass concentration of 28-32%, ethanol aqueous solution with a mass concentration of 48-52%, ethanol aqueous solution with a mass concentration of 68-72%, and ethanol aqueous solution with a mass concentration of 78-82%; after the gradient elution step, the ethanol aqueous solution with a mass concentration of 28-32% is concentrated to obtain a flavonoid-rich extract; the ethanol aqueous solution with a mass concentration of 48-52% is concentrated to obtain a saponin-rich extract. This invention utilizes a macroporous resin column formed by a specific ratio of D101 macroporous resin and HPD-600 macroporous resin, and optimizes the elution gradient. Fractional elution with 10%-80% ethanol aqueous solution further precisely separates flavonoid-rich extracts (30% ethanol aqueous solution fraction) and saponin-rich extracts (50% ethanol aqueous solution fraction). The extraction efficiency and purity of both flavonoid-rich and saponin-rich extracts are high.

[0017] The method for separating flavonoids and saponins from jujube leaves provided by this invention is green and efficient. It uses ultrasonic pretreatment to shorten the extraction time, reduce energy consumption, and avoid degradation of heat-sensitive components. At the same time, it has a high component recovery rate, a short process cycle, and the macroporous resin can be reused multiple times, resulting in a long service life. Detailed Implementation

[0018] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0019] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products. Example 1

[0020] This embodiment provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 40-mesh sieve to obtain jujube leaf powder, mix 30g of jujube leaf powder with 600mL of water, and perform the first ultrasonic treatment at a frequency of 40 kHz, an ultrasonic power of 300w, an ultrasonic temperature controlled at 50℃, and an ultrasonic time of 1h. Then, perform the first hot reflux extraction at 85℃ for 2h, and filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 600 mL of ethanol aqueous solution with a mass concentration of 50%. The solid residue and ethanol aqueous solution are subjected to a second ultrasonic treatment with an ultrasonic frequency of 40 kHz, an ultrasonic power of 300 W, an ultrasonic temperature controlled at 50 °C, and an ultrasonic time of 1 h. Then, a second hot reflux extraction is performed at 75 °C for 2 h to obtain extract B. 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 60℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting 2 BV with deionized water at a flow rate of 2 BV / h to remove polysaccharides and proteins, then eluting 2 BV with 10% ethanol aqueous solution at a flow rate of 2 BV / h to remove polar impurities, and then eluting 3 BV with 30% ethanol aqueous solution at a flow rate of 3 BV / h. The resin was eluted with a 50% ethanol aqueous solution at a flow rate of 3 BV / h for 3 BV, and a 70% ethanol aqueous solution at a flow rate of 2 BV / h for 2 BV. The resin was then regenerated, and an 80% ethanol aqueous solution was eluted with a flow rate of 1 BV / h for 1 BV. The resin was thoroughly cleaned. The eluent of a 30% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 6.071% and a purity of 79.6%. The eluent of a 50% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 1.996% and a purity of 78.2%. Example 2

[0021] This embodiment provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 20-mesh sieve to obtain jujube leaf powder, mix 200g of jujube leaf powder with 4L of water, and perform the first ultrasonic treatment at a frequency of 40 kHz, an ultrasonic power of 500w, an ultrasonic temperature controlled at 50℃, and an ultrasonic time of 1h. Then, perform the first hot reflux extraction at 85℃ for 2h, and filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 4L of ethanol aqueous solution with a mass concentration of 50%. The solid residue and ethanol aqueous solution are subjected to a second ultrasonic treatment with an ultrasonic frequency of 40 kHz, an ultrasonic power of 500w, an ultrasonic temperature controlled at 50℃, and an ultrasonic time of 1h. Then, a second hot reflux extraction is performed at 75℃ for 2h to obtain extract B. 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 60℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting with deionized water at a flow rate of 2.5 BV / h for 2 BV to remove polysaccharides and proteins, and then eluting with 10% ethanol aqueous solution at a flow rate of 2.5 BV / h for 2 BV. BV was used to remove polar impurities. Elution was performed using a 30% ethanol aqueous solution at a flow rate of 3.5 BV / h for 3 BV, followed by a 50% ethanol aqueous solution at a flow rate of 3.5 BV / h for 3 BV, and a 70% ethanol aqueous solution at a flow rate of 2 BV / h for 2 BV. The resin was then regenerated, and eluted with an 80% ethanol aqueous solution at a flow rate of 3.5 BV / h for 1 BV. The resin was thoroughly cleaned. The 30% ethanol aqueous solution eluent was collected and concentrated under reduced pressure at 60°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 7.17% and a purity of 83.1%. The 50% ethanol aqueous solution eluent was collected and concentrated under reduced pressure at 60°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 2.68% and a purity of 80.7%. Example 3

[0022] This embodiment provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 40-mesh sieve to obtain jujube leaf powder, mix 30g of jujube leaf powder with 600mL of water, and perform the first ultrasonic treatment at a frequency of 40 kHz, an ultrasonic power of 400w, an ultrasonic temperature controlled at 50℃, and an ultrasonic time of 1h. Then, perform the first hot reflux extraction at 85℃ for 2h, and filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 600 mL of ethanol aqueous solution with a mass concentration of 50%. The solid residue and ethanol aqueous solution are subjected to a second ultrasonic treatment with an ultrasonic frequency of 40 kHz, an ultrasonic power of 400 W, an ultrasonic temperature controlled at 50 °C, and an ultrasonic time of 1 h. Then, a second hot reflux extraction is performed at 75 °C for 2 h to obtain extract B. 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 50℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting 2 BV with deionized water at a flow rate of 1 BV / h to remove polysaccharides and proteins, then eluting 2 BV with 10% ethanol aqueous solution at a flow rate of 1 BV / h to remove polar impurities, and then eluting 3 BV with 30% ethanol aqueous solution at a flow rate of 2 BV / h. The resin was eluted with a 50% ethanol aqueous solution at a flow rate of 2 BV / h for 3 BV, and with a 70% ethanol aqueous solution at a flow rate of 1.5 BV / h for 2 BV. The resin was then regenerated, and with an 80% ethanol aqueous solution at a flow rate of 0.5 BV / h for 1 BV. The resin was thoroughly cleaned. The eluent of a 30% ethanol aqueous solution was collected and concentrated under reduced pressure at 50°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 6.83% and a purity of 80.5%. The eluent of a 50% ethanol aqueous solution was collected and concentrated under reduced pressure at 50°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 2.57% and a purity of 81.3%. Example 4

[0023] This embodiment provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 40-mesh sieve to obtain jujube leaf powder, mix 30g of jujube leaf powder with 600mL of water, and perform the first ultrasonic treatment at a frequency of 40 kHz, an ultrasonic power of 600w, an ultrasonic temperature controlled at 50℃, and an ultrasonic time of 1h. Then, perform the first hot reflux extraction at 80℃ for 2h, and filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 600 mL of ethanol aqueous solution with a mass concentration of 50%. The solid residue and ethanol aqueous solution are subjected to a second ultrasonic treatment with an ultrasonic frequency of 40 kHz, an ultrasonic power of 600 W, an ultrasonic temperature controlled at 50 °C, and an ultrasonic time of 1 h. Then, a second hot reflux extraction is performed at 75 °C for 2 h to obtain extract B. 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 80℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting 2 BV with deionized water at a flow rate of 3 BV / h to remove polysaccharides and proteins, then eluting 2 BV with 10% ethanol aqueous solution at a flow rate of 3 BV / h to remove polar impurities, and then eluting 3 BV with 30% ethanol aqueous solution at a flow rate of 4 BV / h. The resin was eluted with a 50% ethanol aqueous solution at a flow rate of 4 BV / h for 3 BV, and with a 70% ethanol aqueous solution at a flow rate of 2.5 BV / h for 2 BV. The resin was then regenerated, and with an 80% ethanol aqueous solution at a flow rate of 1.5 BV / h for 1 BV. The resin was thoroughly cleaned. The eluent of a 30% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 6.33% and a purity of 81.2%. The eluent of a 50% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 2.47% and a purity of 80.7%.

[0024] Comparative Example 1 This comparative example provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 40-mesh sieve to obtain jujube leaf powder, mix 30g of jujube leaf powder with 600mL of water, and perform a first hot reflux extraction at 85℃ for 2h. Filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 600 mL of water, and then perform a second hot reflux extraction at 85 °C for 2 h to obtain extract B; 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 60℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting 2 BV with deionized water at a flow rate of 2 BV / h to remove polysaccharides and proteins, then eluting 2 BV with 10% ethanol aqueous solution at a flow rate of 2 BV / h to remove polar impurities, and then eluting 3 BV with 30% ethanol aqueous solution at a flow rate of 3 BV / h. The resin was eluted with a 50% ethanol aqueous solution at a flow rate of 3 BV / h for 3 BV, and a 70% ethanol aqueous solution at a flow rate of 2 BV / h for 2 BV. The resin was then regenerated, and an 80% ethanol aqueous solution was eluted with a flow rate of 1 BV / h for 1 BV. The resin was thoroughly cleaned. The eluent of a 30% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 4.35% and a purity of 62.8%. The eluent of a 50% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 1.02% and a purity of 56.3%.

[0025] Comparative Example 2 This embodiment provides a method for separating flavonoids and saponins from jujube leaves, including the following steps: 1) Take dried jujube leaves, crush them through a 40-mesh sieve to obtain jujube leaf powder, mix 30g of jujube leaf powder with 600mL of ethanol aqueous solution with a mass concentration of 70%, and then perform the first hot reflux extraction at 85℃ for 2h. Filter to separate extract A and solid residue. 2) Mix the solid residue obtained in step 1) with 600 mL of ethanol aqueous solution with a mass concentration of 70%, and then perform a second hot reflux extraction at 75°C for 2 h to separate and obtain extract B; 3) Mix extract A obtained in step 1) and extract B obtained in step 2), concentrate under reduced pressure at 60℃ to remove ethanol, forming 180mL of concentrate. Then, use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1. Soak the macroporous resin column in 95% ethanol aqueous solution for more than 24 hours, wash with deionized water until there is no ethanol odor, then soak in 5% sodium hydroxide solution for 24 hours, and then soak in deionized water until neutral (pH test paper test); finally, soak in 5% hydrochloric acid for 24 hours, and wash with deionized water until neutral. Then, load the concentrate into the macroporous resin column at a flow rate of 2 BV / h for gradient elution, including eluting 2 BV with deionized water at a flow rate of 2 BV / h to remove polysaccharides and proteins, then eluting 2 BV with 10% ethanol aqueous solution at a flow rate of 2 BV / h to remove polar impurities, and then eluting 3 BV with 30% ethanol aqueous solution at a flow rate of 3 BV / h. The resin was eluted with a 50% ethanol aqueous solution at a flow rate of 3 BV / h for 3 BV, and a 70% ethanol aqueous solution at a flow rate of 2 BV / h for 2 BV. The resin was then regenerated, and an 80% ethanol aqueous solution was eluted with a flow rate of 1 BV / h for 1 BV. The resin was thoroughly cleaned. The eluent of a 30% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a pale yellow flavonoid-rich extract powder with a yield of 4.07% and a purity of 58.6%. The eluent of a 50% ethanol aqueous solution was collected and concentrated under reduced pressure at 60°C to obtain a brownish-yellow saponin-rich extract powder with a yield of 1.63% and a purity of 64.2%. Some saponins were lost due to precipitation caused by the high ethanol concentration.

[0026] Comparative Example 3 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1" is replaced with "a macroporous resin column formed using HPD-600 macroporous resin".

[0027] Comparative Example 4 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin at a mass ratio of 1:1" is replaced with "a macroporous resin column formed using D101 macroporous resin".

[0028] Comparative Example 5 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin in a mass ratio of 1:1" is replaced with "a macroporous resin column formed by combining D101 macroporous resin and AB-8 macroporous resin in a mass ratio of 1:1".

[0029] Comparative Example 6 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin in a mass ratio of 1:1" is replaced with "a macroporous resin column formed by combining AB-8 macroporous resin and HPD-600 macroporous resin in a mass ratio of 1:1".

[0030] Comparative Example 7 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin in a mass ratio of 1:1" is replaced with "a macroporous resin column formed by combining D101 macroporous resin and polyamide resin in a mass ratio of 1:1".

[0031] Comparative Example 8 This comparative example provides a method for separating flavonoids and saponins from jujube leaves. The only difference from Example 1 is that in step 3), "a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin in a mass ratio of 1:1" is replaced with "a macroporous resin column formed by combining polyamide resin and HPD-600 macroporous resin in a mass ratio of 1:1".

[0032] Test Example 1 The pale yellow flavonoid-rich extract powders obtained in Examples 1-4 or Comparative Examples 1-8 were subjected to flavonoid purity and content testing, and the saponin-rich extract powders obtained in Examples 1-4 or Comparative Examples 1-8 were subjected to saponin purity and content testing. The specific testing methods are as follows: Flavonoid testing: (1) Accurately weigh 10.0 mg of rutin standard, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and prepare a stock solution of 1.0 mg / mL. Accurately pipette 0.1, 0.2, 0.4, 0.6, and 0.8 mL of the stock solution into 10 mL volumetric flasks respectively; then add 0.5 mL of 5 wt% NaNO2 solution to each volumetric flask, shake well, let stand for 6 min, then add 0.5 mL of 10 wt% Al(NO3)3 solution, let stand for 6 min, and finally add 4 mL of 4 wt% NaOH solution, dilute to the mark with 70 wt% ethanol, shake well, and let stand for 15 min (NaNO2-Al(NO3)3 colorimetric method). Prepare a blank solution without rutin using the same method (using 70 wt% ethanol instead of the stock solution). The absorbance was measured at a wavelength of 510 nm. A standard curve was plotted with rutin concentration (X, mg / mL) on the x-axis and absorbance (Y) on the y-axis. The slope of the standard curve was obtained as k (linear range: 0.01–0.08 mg / mL, R0). 2 ≥0.999).

[0033] (2) Accurately weigh 10 mg of the flavonoid-rich extract obtained in Examples 1-4 or Comparative Examples 1-9 (3 parallel samples per group), place them in 10 mL volumetric flasks, add methanol, sonicate to dissolve and dilute to the mark, and shake well. Take 1.0 mL of the above solution and place it in 10 mL volumetric flasks. After processing according to the method in step (1) above (NaNO2-Al(NO3)3 colorimetric method), measure the absorbance at a wavelength of 510 nm. The blank solution without rutin is prepared according to the method in step (1). The flavonoid content (calculated as rutin) is calculated using the external standard two-point method, thereby calculating the yield and purity of flavonoids. The flavonoid concentration in the sample is C. 样品 =A 样品 / k, in units of (ml / mg), where A 样品 ρ represents the absorbance of the sample, and k represents the slope of the standard curve. Flavonoid purity (%) = CVD / W × 100%, where C: flavonoid concentration calculated from the standard curve (mg / ml), V: final volume (mL), D: dilution factor, and W: sample mass (mg). Flavonoid yield = (mass of flavonoid-rich extract powder × flavonoid purity / mass of jujube leaf powder) × 100%. The test results are shown in Table 1.

[0034] Saponin detection: The test was performed using HPLC, and the test conditions were as follows: (1) Chromatographic conditions: Chromatographic column: C18 reversed-phase column (250 mm x 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) - 0.1% aqueous phosphoric acid solution (B); Gradient elution: 0-15 min: 25%-40% A, 15-25 min: 40%-55% A; 25-30 min: 55%→70% A (washing column), 30-35 min: 25% A (equilibration); Flow rate: 1.0 mL / min; Detection wavelength: 203 nm (terminal absorption of soap-like substances); Column temperature: 30℃; Injection volume: 10 μL; (2) Preparation of reference solution: ① Standard curve of jujuboside A: Accurately weigh 10.0 mg of jujuboside A reference standard, dissolve it in methanol and dilute to 10 mL (1.0 mg / mL). Serially dilute to 5, 10, 20, 40, and 80 μg / mL, inject into HPLC, and record the peak area with concentration (μg / mL) on the x-axis (X) and peak area on the y-axis. Fit a standard curve (R ≥ 0.999).

[0035] ②The standard curve of jujube seed saponin B was prepared by preparing a series of concentration solutions of jujube seed saponin B using the same method, and a standard curve was established.

[0036] (3) Sample pretreatment Accurately weigh 10 mg of the brownish-yellow powder obtained from the 50% ethanol elution, dissolve it in methanol and bring the volume to 10 mL, sonicate for 10 min, filter through a 0.45 μm filter membrane, and prepare a blank solution (methanol) in the same way.

[0037] (4) Inject the sample solution and record the peak areas of jujube seed saponins A and B (the retention time must be consistent with the reference standard).

[0038] The purity and yield of saponins were calculated using the following methods: The yield of jujube seed saponin A = (sample peak area corresponding to A concentration × fixed volume) / jujube leaf mass × 100%; the yield of jujube seed saponin B = (sample peak area corresponding to B concentration × fixed volume) / jujube leaf mass × 100%; the total saponin yield (%) = A yield + B yield; the total saponin purity (%) = (peak area of ​​saponins A and B / total peak area) × 100%. The test results are shown in Table 1.

[0039] Table 1

[0040] The yield and purity of the target components extracted by the method of the present invention are significantly higher than those of Comparative Example 1 (single water extraction) or Comparative Example 2 (single alcohol extraction) and other comparative examples. After resin purification, the yield of the target components extracted by the method of the present invention is more than 58% higher than that of Comparative Example 1, more than 49% higher than that of Comparative Example 2, the purity of flavonoids is more than 26% higher than that of Comparative Example 1, and the purity of saponins is more than 38% higher than that of Comparative Example 1.

[0041] Test Example 2 The method of Example 2 was repeated for 5 batches to verify stability. The stability test was passed when the flavonoid yield was ≥6%, the flavonoid purity was ≥75%, the saponin yield was ≥1.8%, and the saponin purity was ≥75% in all 5 repeated experiments. The results are shown in Table 2. All 5 experiments met the requirements and the stability test was passed.

[0042] Table 2

[0043] Test Example 3 Animal sleep activity verification: (1) Experimental materials: The flavonoid-rich extract powder obtained in Example 2, the saponin-rich extract powder obtained in Example 2, and the flavonoid-rich extract powder and saponin-rich extract powder obtained in Example 2 or Comparative Examples 1-2 were mixed to form the mixed extracts of Example 2 or Comparative Examples 1-2. A solution of 0.005 g / mL was prepared with physiological saline as the experimental group material. Diazepam (1 mg / kg) was prepared with physiological saline as a solution of 0.005 g / mL as the sample control material. 0.005% physiological saline solution was used as the blank control material. (2) Experimental animals: ICR mice, male, 120 mice, weighing 18-22g, were fed with ordinary feed under constant temperature conditions without special pathogens, at a temperature of (23±2)℃ and a relative humidity of 50±10%, and were housed in individual cages. (3) Experimental grouping test: After one week of acclimatization in the animal room, the mice were randomly divided into 12 groups of 10 mice each. Group 1 was the blank control group, which was administered 0.005% saline solution by gavage at a dose of 10 ml / kg based on the mice's body weight. Group 2 was the sample control group, which was administered 0.005% diazepam solution by gavage at a dose of 10 ml / kg based on the mice's body weight. Group 3 was administered 0.005% diazepam solution extracted in Example 2. The extract solution was administered orally to mice at a dosage of 10 ml / kg based on mouse body weight. Group 4 received a 0.005% solution of the flavonoid-rich extract from Example 2, administered orally at a dosage of 5 ml / kg based on mouse body weight. Group 5 received a 0.005% solution of the saponin-rich extract from Example 2, administered orally at a dosage of 10 ml / kg based on mouse body weight. Group 6 received a 0.005% solution of the saponin-rich extract from Example 2, administered orally. The dosage was 5 ml / kg based on mouse body weight; Group 7 was administered a 0.005% mixed extract solution from Example 2 via gavage, at a dosage of 10 ml / kg based on mouse body weight; Group 8 was administered a 0.005% mixed extract solution from Example 2 via gavage, at a dosage of 5 ml / kg based on mouse body weight; Group 9 was administered a 0.005% mixed extract solution from Comparative Example 1 via gavage, at a dosage of 10 ml / kg based on mouse body weight. Group 10 was administered a 0.005% concentration of the mixed extract solution from Comparative Example 1 via gavage at a dose of 5 ml / kg based on mouse body weight; Group 11 was administered a 0.005% concentration of the mixed extract solution from Comparative Example 2 via gavage at a dose of 10 ml / kg based on mouse body weight; Group 12 was administered a 0.005% concentration of the mixed extract solution from Comparative Example 2 via gavage at a dose of 5 ml / kg based on mouse body weight; each group was administered the medication once daily.

[0044] Mice in each group were allowed free access to water and food. After 14 days of continuous gavage, 30 minutes after the last administration, they were injected intraperitoneally with sodium pentobarbital at a dose of 30 mg / kg (the mice were fasted and not given water for 12 hours before the intraperitoneal injection). The sleep latency and sleep duration of the mice were observed and recorded.

[0045] Sleep assessment criteria: Sleep was indicated by the disappearance of the righting reflex. When mice were placed in a dorsal position, they should be able to immediately right themselves. If they could not right themselves within one minute, the righting reflex was considered to have disappeared, and they had entered sleep. Sleep latency refers to the time from administration of sodium pentobarbital to the disappearance of the righting reflex. The recovery of the righting reflex is considered wakefulness of the mouse, and the time from the disappearance to the recovery of the righting reflex is considered sleep time. The experimental results are shown in Table 3: Table 3

[0046] The sleep latency data of groups 3 to 8 in this test case were significantly shorter than those of groups 1, 2, and 9 to 12, and the sleep duration data were also significantly longer than those of groups 1, 2, and 9 to 12. This proves that the jujube bud and leaf extract extracted by the method of this invention can shorten the sleep latency of mice, prolong sleep time, and has a clear sedative-hypnotic effect.

[0047] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for separating flavonoids and saponins from jujube leaves, characterized in that, Includes the following steps: 1) Mix jujube leaf powder with water, perform a first ultrasonic treatment, and then perform a first hot reflux extraction to separate extract A and solid residue; 2) Mix the solid residue obtained in step 1) with the alcohol-water solution, subject it to a second ultrasonic treatment, and then perform a second hot reflux extraction to obtain extract B; 3) Mix the extract A obtained in step 1) and the extract B obtained in step 2), concentrate to remove ethanol to form a concentrate, and then use a macroporous resin column formed by combining D101 macroporous resin and HPD-600 macroporous resin to perform gradient elution of the concentrate to obtain the flavonoid-rich extract and saponin-rich extract.

2. The preparation method of separating flavonoids and saponins from jujube leaves according to claim 1, characterized in that, In step 3), the mass ratio of D101 macroporous resin to HPD-600 macroporous resin in the macroporous resin column formed by the composite of D101 macroporous resin and HPD-600 macroporous resin is 1:(0.5-1.5). And / or, the gradient elution concentrate step includes: loading the concentrate into a macroporous resin column, and then eluting with water, an ethanol aqueous solution with a mass concentration of 8-12%, an ethanol aqueous solution with a mass concentration of 28-32%, an ethanol aqueous solution with a mass concentration of 48-52%, an ethanol aqueous solution with a mass concentration of 68-72%, and an ethanol aqueous solution with a mass concentration of 78-82%. After the gradient elution step is completed, the eluent with an ethanol aqueous solution of 28-32% by mass is concentrated to obtain a flavonoid-rich extract; the eluent with an ethanol aqueous solution of 48-52% by mass is concentrated to obtain a saponin-rich extract.

3. The preparation method of separating flavonoids and saponins from jujube leaves according to claim 2, characterized in that, The elution flow rate of the water elution is 1-3 BV / h, and the elution volume is 1-3 column volumes; The elution flow rate of the ethanol aqueous solution with a mass concentration of 8-12% is 1-3 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate for the ethanol aqueous solution with a mass concentration of 28-32% is 1.2-4 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate of the ethanol aqueous solution with a mass concentration of 48-52% is 2-4 BV / h, and the elution volume is 1-4 column volumes. The elution flow rate of the ethanol aqueous solution with an ethanol mass concentration of 68-72% is 1.5-2.5 BV / h, and the elution volume is 1-3 column volumes. The elution flow rate of the ethanol aqueous solution with a mass concentration of 78-82% is 0.5-1.5 BV / h, and the elution volume is 1-2 column volumes.

4. The preparation method of separating flavonoids and saponins from jujube leaves according to any one of claims 1-3, characterized in that, In step 3), the macroporous resin column formed by the composite of D101 macroporous resin and HPD-600 macroporous resin is soaked in alcohol solution and washed until there is no alcohol odor, soaked in alkaline solution and washed until neutral, and soaked in acid solution and washed until neutral.

5. The preparation method of separating flavonoids and saponins from jujube leaves according to claim 4, characterized in that, The alcohol concentration in the alcohol solution is 90-99% by mass. The alcohol solution is selected from aqueous ethanol solution; And / or, the mass concentration of alkali in the alkaline solution is 3-8%; The alkaline solution is selected from an aqueous sodium hydroxide solution; And / or, the mass concentration of acid in the acid solution is 3-8%; The acid solution is selected from hydrochloric acid aqueous solution and / or phosphoric acid aqueous solution; And / or, the soaking time in the alcohol solution is 24-72 hours; And / or, the immersion time in the alkaline solution is 12-36 hours; And / or, the acid solution soaking time is 12-36 hours.

6. The preparation method of separating flavonoids and saponins from jujube leaves according to any one of claims 1-5, characterized in that, The jujube leaf powder mentioned in step 1) is jujube leaf powder that has passed through a 20-40 mesh sieve; The ratio of jujube leaf powder to water in step 1) is 1:(18-22), with units of g / mL; The ultrasonic power of the first ultrasonic treatment is 300-600W, the ultrasonic treatment temperature is 48-52℃, and the ultrasonic treatment time is 0.5-2h. The first hot reflux extraction temperature is 70-90℃, and the first hot reflux extraction time is 1.5-2.5h.

7. The preparation method of separating flavonoids and saponins from jujube leaves according to any one of claims 1-6, characterized in that, The ratio of the solid residue to the ethanol-water solution in step 2) is (0.5-1.1):(18-22), in g / mL. The alcohol-water solution has an alcohol concentration of 48-52% by mass. The aqueous alcohol solution is selected from aqueous ethanol solution; The ultrasonic power of the second ultrasonic treatment is 300-600W, the ultrasonic treatment temperature is 48-52℃, and the ultrasonic treatment time is 0.5-2h. The second hot reflux extraction temperature is 70-90℃, and the second hot reflux extraction time is 1.5-2.5h; The concentration described in step 3) is vacuum concentration, which can be selected, and the concentration temperature is 50-80℃.

8. A flavonoid-rich extract, characterized in that, It is obtained by the method for separating flavonoids and saponins from jujube leaves according to any one of claims 1-7.

9. A saponin-rich extract, characterized in that, It is obtained by the method for separating flavonoids and saponins from jujube leaves according to any one of claims 1-7.

10. The method for separating flavonoids and saponins from jujube leaves according to claim 8 and / or according to claim 9, in the preparation of a sleep aid drug.

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