Method for extracting and purifying chlorogenic acid and flavone from folium cortex eucommiae

Abstract

The invention discloses a method for extracting and purifying chlorogenic acid and flavone from folium cortex eucommiae, which comprises the following steps: step 1, carrying out ultrasonic extraction on folium cortex eucommiae by using an ethanol water solution to obtain a folium cortex eucommiae extracting solution; step 2, performing rough filtration on the eucommia ulmoides extracting solution obtained in the step 1 by using filter paper to obtain first penetrating fluid; step 3, performing microfiltration on the first penetrating fluid obtained in the step 2 by using a ceramic microfiltration membrane to obtain second penetrating fluid; step 4, performing ultrafiltration on the second penetrating fluid obtained in the step 3 by using a ceramic ultrafiltration membrane to obtain third penetrating fluid; and 5, carrying out rotary evaporation on the third penetrating fluid obtained in the step 4, collecting a concentrated solution, and drying to obtain a mixture of chlorogenic acid and flavone. By using the method provided by the invention, the yield of chlorogenic acid can reach 90% or more, and the use of the ethanol aqueous solution extractant makes the membrane module not easy to block, shortens the cleaning and maintenance period of the membrane module, reduces the process operation cost, and improves the process operation stability.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and in particular to a method for extracting and purifying chlorogenic acid and flavonoids from eucommia ulmoides leaves. Background Art

[0002] Eucommia ulmoides is a unique medicinal tree species with abundant resources in my country. It has the effects of nourishing the liver and kidneys, strengthening bones and muscles, and lowering blood pressure. It is a high-quality raw material for extracting natural active products, among which small molecules such as chlorogenic acid and flavonoids have obvious medicinal value. Traditional Chinese medicine's demand for Eucommia ulmoides mainly lies in Eucommia ulmoides bark, but it has problems such as a long growth cycle (over 12 years) and poor survival rate of trees after peeling. Eucommia ulmoides leaves and Eucommia ulmoides bark have similar chemical compositions, and Eucommia ulmoides leaves have a short growth cycle, high yield, and are relatively easy to harvest. In 2017, Eucommia ulmoides leaves were included in the list of medicinal and edible plants, and there is great potential for replacing Eucommia ulmoides bark with Eucommia ulmoides leaves as medicine.

[0003] The current production process of Eucommia products, the filtration and concentration process, uses traditional screen filtration and vacuum concentration. The process often faces problems such as unsatisfactory filtration effect, easy impact on product quality stability, and high energy consumption for vacuum concentration. Membrane separation technology has the advantages of high separation efficiency, no secondary pollution, low energy consumption, small footprint, and simple operation. It has been widely used in the field of biomedicine in recent years. However, the widespread problem of membrane pollution and the high temperature and solvent resistance of the membrane have limited the large-scale application of membrane technology to a certain extent. Traditional organic membranes cannot meet the stringent requirements of the traditional Chinese medicine system. Ceramic membranes have the advantages of narrow pore size distribution, high temperature resistance, solvent resistance, and easy cleaning, which can effectively cope with the high temperature and organic solvent environment of the traditional Chinese medicine system. Our team previously proposed a double membrane method to separate the crude Eucommia extract (CN 114409542B), in which fat-soluble impurities are removed by the solvent phase, and solid suspended matter and emulsified phase are removed by membrane separation, which effectively improves the purity of chlorogenic acid. Ethyl acetate is used for extraction, and the stable flux of the aqueous phase is 73L·m -2 ·h -1 The stable flux of oil phase is 34 L·m -2 ·h -1 The crude Eucommia ulmoides extract available on the market has unstable active ingredient content and an unknown production process. Furthermore, existing technologies, which use complex extraction methods to obtain active ingredients such as chlorogenic acid and flavonoids, have problems with efficacy stability and storage. Summary of the Invention

[0004] The purpose of the present invention is to address the problems existing in the prior art and provide a method for extracting and purifying chlorogenic acid and flavonoids from Eucommia ulmoides leaves. The method combines alcohol solution extraction and membrane separation, and directly extracts the effective ingredients of Eucommia ulmoides using ethanol aqueous solution, replacing water extraction and alcohol precipitation to reduce extraction time. The ethanol aqueous solution is used to remove some insoluble impurities, while reducing the dissolution of water-soluble polysaccharides to reduce membrane pollution in subsequent membrane processes. The Eucommia ulmoides leaf extract is efficiently clarified using a ceramic microfiltration membrane, and large molecular non-pharmaceutical components such as starch and protein are further removed using a ceramic ultrafiltration membrane. The product is obtained by rotary evaporation and freeze drying.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A method for extracting and purifying chlorogenic acid and flavonoids from Eucommia ulmoides leaves comprises the following steps:

[0007] Step 1, ultrasonically extracting the Eucommia ulmoides leaves with an ethanol aqueous solution to obtain an Eucommia ulmoides extract;

[0008] Step 2, coarsely filtering the Eucommia ulmoides extract obtained in step 1 with filter paper to obtain a first permeate;

[0009] Step 3, microfiltration of the first permeate obtained in step 2 using a ceramic microfiltration membrane to obtain a second permeate;

[0010] Step 4, ultrafiltration of the second permeate obtained in step 3 using a ceramic ultrafiltration membrane to obtain a third permeate;

[0011] Step 5, rotary evaporating the third permeate obtained in step 4, collecting the concentrated solution, and drying it to obtain a mixture of chlorogenic acid and flavonoids.

[0012] In step 1, the mass fraction of the ethanol aqueous solution is 30-95%, preferably 50%; the mass ratio of the Eucommia ulmoides leaves to the ethanol aqueous solution is 1:(10-200), preferably 1:20 or 1:200.

[0013] In step 1, the frequency of the ultrasound is 50 to 55 kHz, preferably 53 kHz, and the time is 1 to 2 hours; the ultrasonic extraction is carried out at a temperature of 40 to 100° C., preferably 60° C.; the number of ultrasonic extractions is 2 to 3 times.

[0014] In step 2, the filter paper is a rapid qualitative filter paper with a pore size of 20 to 80 μm.

[0015] In step 3, the average pore size of the ceramic microfiltration membrane is 100 to 500 nm, preferably 100 nm;

[0016] In step 3, the operating pressure during the microfiltration process is 1-5 bar, preferably 2-3 bar, the operating temperature is 20-80°C, preferably 40-50°C, the filtration method is cross-flow filtration, and the membrane surface flow rate is 0.5-5 m / s, preferably 3-5 m·s -1 .

[0017] In step 4, the average pore size of the ceramic ultrafiltration membrane is 2 to 20 nm.

[0018] In step 4, the operating pressure during the ultrafiltration process is 1-5 bar, preferably 2-3 bar, the operating temperature is 20-80°C, preferably 40-50°C, the filtration method is cross-flow filtration, and the membrane surface flow rate is 0.5-5 m / s, preferably 3-5 m·s -1 .

[0019] The eucommia extract is subjected to membrane separation by a ceramic microfiltration membrane and a ceramic ultrafiltration membrane to remove impurities therein, including some macromolecular proteins, polysaccharides, starch, alkaloids, tannins, lipids, organelles, etc., and a clear liquid containing chlorogenic acid and flavonoids is obtained in the permeate after microfiltration.

[0020] The raw material eucommia leaves contain 1-5 wt% of chlorogenic acid and 0.3-2 wt% of flavonoids.

[0021] Chlorogenic acid, the active ingredient in Eucommia ulmoides, is easily soluble in hot water and ethanol; whereas flavonoids are generally poorly soluble or insoluble in water, flavonoids readily combine with sugars to form flavonoid glycosides, which are readily soluble in highly polar solvents such as water, methanol, and ethanol. Taking both active ingredients into consideration, the present invention employs an ethanol-water solution as the extraction solvent for chlorogenic acid and flavonoids, the active ingredients in Eucommia ulmoides. This reduces the chance of the extract becoming moldy and facilitates industrial production. However, when the ethanol content in the extractant exceeds 75 wt%, proteins and polysaccharides begin to precipitate, and chlorogenic acid and flavonoids in Eucommia ulmoides easily aggregate with proteins and polysaccharides. Small molecules such as chlorogenic acid and flavonoids can be entrained by larger molecules such as proteins and polysaccharides, and settle together, resulting in loss of the product's active ingredients. Therefore, the ethanol concentration of the extraction solvent needs to be carefully considered.

[0022] The ethanol aqueous solution extraction process is coupled with the ceramic membrane separation process to explore the compatibility thereof. It is possible to replace the traditional water extraction and alcohol precipitation process, achieve the goal of improving quality and efficiency, energy conservation and emission reduction, and promote the modernization of the Chinese medicine industry. On this basis, the present invention uses the Eucommia ulmoides leaf, which is cheap and highly reproducible, as raw material and is extracted using ethanol aqueous solution as solvent, replacing the traditional water extraction and alcohol precipitation process. It is possible to reduce the dissolution of water-soluble impurities to a certain extent, improve effective component extraction efficiency and process stability, and reduce time cost and raw material cost. When using ceramic membrane to process the Eucommia ulmoides water extract, the macromolecular substances such as water-soluble polysaccharides and proteins are easily polymerized to cause the formation of membrane pollution, so that membrane flux is reduced. The content of macromolecular impurities in the alcohol extract of Eucommia ulmoides leaves is significantly lower than that in the water extract. The extraction rate of effective ingredients in Eucommia ulmoides leaves by 50wt% ethanol aqueous solution is significantly higher, and it is well compatible with membrane technology. When the alcohol extract of Eucommia ulmoides leaves is treated by a composite ceramic membrane process of microfiltration clarification and ultrafiltration refinement, the stable flux of the membrane is significantly improved, the turbidity removal rate is close to 100%, and the product purity and yield are significantly improved.

[0023] Beneficial effects:

[0024] The extraction of chlorogenic acid and flavonoids, the active ingredients in Eucommia ulmoides leaves, using a 50% by mass ethanol aqueous solution as an extractant can reduce the dissolution of water-soluble impurities (polysaccharides, proteins, etc.) while removing insoluble impurities, and extracting active ingredients such as chlorogenic acid and flavonoids as much as possible. The removal of impurities such as polysaccharides and proteins is beneficial to the subsequent membrane separation process. After the extract is treated with a ceramic microfiltration membrane, 50% of the impurities can be removed, and the yield of chlorogenic acid can reach more than 90%, the permeate is clear and transparent, and the turbidity removal rate is close to 100%. The purity of chlorogenic acid is greatly improved after treatment with a ceramic ultrafiltration membrane. The method of the present invention is simple, fast, efficient, and low-cost. It has reached a cooperation with Jiangxi Puzheng Pharmaceutical. For large varieties of traditional Chinese medicine such as whole Eucommia capsules, a new membrane-based Chinese medicine extract refining and concentration process with "microfiltration-ultrafiltration-nanofiltration" multi-membrane integration as the core has been developed, and on-site pilot and process verification have been completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0026] Figure 1 It is a schematic diagram of the process flow of the present invention;

[0027] Figure 2 This is a schematic diagram of the ceramic membrane cross-flow filtration process of the present invention;

[0028] Figure 3 The statistical graph of membrane stable flux after filtering Eucommia ulmoides leaf extract through ceramic membranes with different pore sizes for 1 hour in Comparative Example 4;

[0029] Figure 4 This is a statistical diagram of membrane permeation flux during the filtration of Eucommia ulmoides extract by ceramic microfiltration membrane in Example 5. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0031] Specific techniques or conditions not specified in the examples were carried out according to those described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer specified were all conventional products available through regular channels.

[0032] The content of chlorogenic acid and flavonoids in the following examples was detected by HPLC under the following chromatographic conditions: chromatographic column: Waters XBridge BEH C18 (250×4.6 mm, 5 μm); mobile phase: methanol (A) and 0.1% glacial acetic acid solution (B); gradient elution (0-25 min, 5% A→95% A; 25-26 min, 95% A→95% A; 26-36 min, 95% A→5% A), flow rate: 0.3 mL / min; column temperature: 25°C; detection wavelength: 350 nm; injection volume: 3 μL.

[0033] The process flow diagram of the present invention is as follows Figure 1 shown.

[0034] Example 1

[0035] A certain amount of dried Eucommia ulmoides leaves were mixed with different solvents (the specific experimental design is shown in Table 1). Ultrasonic extraction was performed at 60°C and 53 kHz for 60 min, repeated twice, to obtain a crude extract. Insoluble matter was then removed using rapid qualitative filter paper and a Büchner funnel to obtain an extract. The chlorogenic acid and flavonoid contents in the extract were determined, and the extraction yields of chlorogenic acid and flavonoids were calculated (the contents of chlorogenic acid and flavonoids in the raw Eucommia ulmoides leaves were 5 wt% and 1.2 wt%, respectively). The results are shown in Table 1. Compared with pure water and other ethanol aqueous solutions, a 50% ethanol aqueous solution as a solvent significantly improved the extraction yield of flavonoids and slightly increased the extraction yield of chlorogenic acid. When the solid-liquid mass ratio was reduced from 1:20 to 1:200, the extraction yields of chlorogenic acid and flavonoids increased slightly. Considering the throughput of the subsequent membrane separation process, a solid-liquid mass ratio of 1:20 was selected as the optimal solid-liquid ratio for extraction.

[0036] Table 1 Extraction rates of chlorogenic acid and flavonoids under different solvents and solid-liquid ratios

[0037]

[0038] Example 2

[0039] The dried Eucommia ulmoides leaves were mixed with a 50% ethanol-water solution (mass ratio of 1:20) and ultrasonically extracted at 60°C for 60 min at 53 kHz to obtain a crude extract of Eucommia ulmoides leaves. The insoluble matter was then removed by coarse filtration using a rapid qualitative filter paper and a Buchner funnel to obtain an extract of Eucommia ulmoides leaves. The extract of Eucommia ulmoides leaves was filtered using a ceramic microfiltration membrane with a pore size of 100 nm. The filtration method was cross-flow filtration. The transmembrane pressure difference during microfiltration was 0.20 MPa, the operating temperature was 50°C, and the membrane surface flow rate was 5 m·s. -1 After 25 hours of operation, the permeation flux of the ceramic microfiltration membrane stabilized at 15 L·m -2 ·h -1 After the microfiltration is completed (after being concentrated 8 times), a microfiltration permeate a is obtained. An equal volume of 50 wt% ethanol aqueous solution is added to the microfiltration concentrate to continue repeated microfiltration. The microfiltration concentrate is microfiltered twice, and the obtained permeate is combined with the microfiltration permeate a to form permeate I. The permeate I is filtered using a ceramic ultrafiltration membrane with a pore size of 10 nm. The filtration method is cross-flow filtration. The transmembrane pressure difference during ultrafiltration is 0.30 MPa, the operating temperature is 50°C, and the membrane surface flow rate is 5 m·s -1 After ultrafiltration, ultrafiltration permeate b is obtained. The ultrafiltration concentrate is also subjected to repeated ultrafiltration (the same method as the microfiltration concentrate) to improve the product yield. The obtained permeate is combined with ultrafiltration permeate b to obtain permeate II. Permeate II is then concentrated by a rotary evaporator and dried in a freeze dryer to obtain chlorogenic acid and flavonoid extracts. The schematic diagram of the ceramic membrane cross-flow filtration process is shown in FIG. Figure 2 shown.

[0040] Testing revealed that the yield of chlorogenic acid in microfiltration permeate A was 82.3%, and the yield of flavonoids was 75.6% (the chlorogenic acid content of the raw Eucommia ulmoides leaves was 4.43 wt%, and the flavonoid content was 0.99 wt%). The yield of chlorogenic acid in permeate I was nearly 95%, and the yield of flavonoids was nearly 90%. After freeze-drying, a crude extract was obtained, in which the combined content of chlorogenic acid and flavonoids was 35.8%.

[0041] Testing revealed that the yield of chlorogenic acid in the ultrafiltration permeate B was 80.6%, and the yield of flavonoids was 73.5% (the chlorogenic acid content of the raw Eucommia ulmoides leaves was 4.43 wt%, and the flavonoid content was 0.99 wt%). The yield of chlorogenic acid in the permeate II was nearly 90%, and the yield of flavonoids was nearly 85%. After freeze-drying, an extract was obtained, in which the chlorogenic acid and flavonoid contents were 50.8%.

[0042] Permeate I was subjected to ethanol precipitation. Ethanol was gradually added to the permeate I until the ethanol content in the mixture of permeate I and ethanol reached 50%, 60%, 75%, 80%, and 95% v / v, respectively. During this process, macromolecular substances in permeate I were gradually precipitated by alcohol. The macromolecular content was analyzed, and the results are shown in Table 2. The alcohol extraction and microfiltration process removed approximately 50% of the impurities. Due to the removal of most starch, mucilage, and some proteins and polysaccharides, the content of pectin and alcohol-soluble components increased slightly, and the content of chlorogenic acid also increased significantly. After membrane separation and clarification, the turbidity removal rate reached approximately 100%, removing substances that cause turbidity in the feed solution and greatly reducing the separation burden of the subsequent ultrafiltration process.

[0043] Table 2 Contents of components in permeate I

[0044]

[0045] Example 3

[0046] Eucommia ulmoides leaves were mixed with a 50% ethanol aqueous solution (mass ratio of material to liquid was 1:20) and ultrasonically extracted at 53kHz for 60 minutes at 50°C to obtain a crude extract of Eucommia ulmoides leaves. The insoluble matter was then coarsely filtered off using a rapid qualitative filter paper and a Buchner funnel to obtain an extract of Eucommia ulmoides leaves. The extract of Eucommia ulmoides leaves was filtered using a ceramic microfiltration membrane with a pore size of 100 nm. The filtration method was cross-flow filtration. The transmembrane pressure difference during microfiltration was about 0.30 MPa, the operating temperature was 50°C, and the membrane surface flow rate was 3 m·s. -1 After the filtration, the microfiltration permeate was obtained, and the microfiltration concentrate was repeatedly filtered (the method was the same as in Example 2), and the permeates were combined to form permeate I; the permeate I was filtered using a ceramic ultrafiltration membrane with a pore size of 20 nm, the filtration method was cross-flow filtration, the transmembrane pressure difference during microfiltration was about 0.30 MPa, the operating temperature was 50°C, and the membrane surface flow rate was 3 m·s -1 After the filtration, the ultrafiltration permeate is obtained, and the ultrafiltration concentrate is repeatedly filtered (the method is the same as that in Example 2), and the permeates are combined to form permeate II; then the permeate II is concentrated by a rotary evaporator and dried by a freeze dryer to obtain an extract.

[0047] The total yield of chlorogenic acid and flavonoids in the permeate I was 88.5%, and the total yield of chlorogenic acid and flavonoids in the permeate II was 84.7%.

[0048] Comparative Example 4

[0049] Eucommia ulmoides leaves were mixed with water (mass ratio of material to liquid was 1:20) and extracted with ultrasonic wave at 60℃ for 120min at 53kHz to obtain a crude extract of Eucommia ulmoides leaves. The insoluble matter was then filtered off using a quick qualitative filter paper and a Buchner funnel to obtain an extract of Eucommia ulmoides leaves. The extract of Eucommia ulmoides leaves was filtered using ceramic membranes with pore sizes of 10, 100, 200 and 500nm, respectively. The membrane pressure difference was about 0.20MPa, the temperature was 50℃ and the membrane surface velocity was 3m·s -1 The permeate is filtered under the operating conditions to obtain the permeate; the permeate is then concentrated by a rotary evaporator and dried by a freeze dryer to obtain the product. Figure 3 As shown in the figure, the stable fluxes of ceramic membranes with different pore sizes after filtration for 1 h were 40.3, 95.4, 44.2 and 42.6 L·m -2 ·h -1 When the membrane pore size is large, smaller contaminants can enter the membrane pores, causing severe pore clogging and contamination, significantly increasing the membrane fouling resistance. Testing the chlorogenic acid and flavonoid content in the permeate showed that a ceramic microfiltration membrane with a pore size of 100 nm had the highest transmittance for chlorogenic acid and flavonoids, at 90.3% and 76.1%, respectively.

[0050] Example 5

[0051] At the pilot site, Eucommia ulmoides leaves were mixed with a 50wt% ethanol aqueous solution at a solid-liquid ratio of 1:10 and extracted twice at 60°C with 53kHz ultrasonic wave extraction for 60 minutes to obtain the Eucommia ulmoides leaf extract. Insoluble matter was coarsely filtered out using rapid qualitative filter paper and a Buchner funnel to obtain the Eucommia ulmoides leaf extract. The extract was filtered using a ceramic microfiltration membrane with a pore size of 100nm. The membrane pressure difference was approximately 0.20MPa, the temperature was 40°C, and the membrane surface velocity was 3m·s. -1 The permeate I was filtered under the operating conditions of , and then the permeate I was concentrated by a rotary evaporator and dried by a freeze dryer to obtain the product. Figure 4 As shown in Figure 2, after running for 1 hour, the permeation flux of the ceramic microfiltration membrane is greater than 60 L·m -2 ·h -1 After 7 hours of operation, the stable permeation flux of the ceramic microfiltration membrane was 24 L·m -2 ·h -1 The permeability of chlorogenic acid and flavonoids was greater than 90%. At this time, the Eucommia ulmoides extract was concentrated 10 times and the turbidity removal rate reached 100%. This shows that the Eucommia ulmoides extract obtained by using ethanol-water as the extraction agent has a much lower clogging effect on the membrane in the subsequent membrane filtration process than the pure water extraction agent.

[0052] The present invention provides a method and concept for extracting and purifying chlorogenic acid and flavonoids from Eucommia ulmoides leaves. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for extracting and purifying chlorogenic acid and flavonoids from Eucommia ulmoides leaves, characterized in that: The steps include: Step 1, ultrasonically extracting the dried Eucommia ulmoides leaves with an ethanol-water solution to obtain an Eucommia ulmoides extract; Step 2, coarsely filtering the Eucommia ulmoides extract obtained in step 1 with filter paper to obtain a first permeate; Step 3, microfiltration of the first permeate obtained in step 2 using a ceramic microfiltration membrane to obtain a second permeate; Step 4, ultrafiltration of the second permeate obtained in step 3 using a ceramic ultrafiltration membrane to obtain a third permeate; Step 5, rotary evaporating the third permeate obtained in step 4, collecting the concentrated solution, and drying it to obtain a mixture of chlorogenic acid and flavonoids.

2. The method according to claim 1, characterized in that In step 1, the mass fraction of the ethanol aqueous solution is 30-95%; the mass ratio of the eucommia leaves to the ethanol aqueous solution is 1:(10-200).

3. The method according to claim 1, characterized in that In step 1, the frequency of the ultrasound is 50-55 kHz, and the time is 1-2 hours; the ultrasonic extraction is carried out at a temperature of 40-100° C.; and the number of ultrasonic extractions is 2-3 times.

4. The method according to claim 1, wherein In step 2, the filter paper is a rapid qualitative filter paper with a pore size of 20 to 80 μm.

5. The method according to claim 1, characterized in that In step 3, the average pore size of the ceramic microfiltration membrane is 100 to 500 nm.

6. The method according to claim 1, characterized in that In step 3, the operating pressure during the microfiltration process is 1 to 5 bar, the operating temperature is 20 to 80° C., and the membrane surface flow rate is 0.5 to 5 m / s.

7. The method according to claim 1, characterized in that In step 4, the average pore size of the ceramic ultrafiltration membrane is 2 to 20 nm.

8. The method according to claim 1, characterized in that In step 4, the operating pressure during the ultrafiltration process is 1 to 5 bar, the operating temperature is 20 to 80° C., and the membrane surface flow rate is 0.5 to 5 m / s.

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