A process for extracting and purifying chlorogenic acid from honeysuckle and purified chlorogenic acid

Abstract

The present invention provides a process for extracting and purifying chlorogenic acid from honeysuckle and the purified chlorogenic acid extracted using this process, belonging to the technical field of natural active ingredient extraction. The present invention comprises extracting a purified crude chlorogenic acid product, purifying the purified crude chlorogenic acid product using an acidic aqueous solution, ethyl acetate, and an alkaline aqueous solution, further purifying the chlorogenic acid using water, ethyl acetate, and water, and finally evaporating and crystallizing the chlorogenic acid using an ethanol-ethyl acetate mixed solvent. The purified chlorogenic acid ultimately produced by the present invention has a purity exceeding 90%. Only three reagents, water, ethanol, and ethyl acetate, are used. Both the ethanol and ethyl acetate used can be reused in a closed-loop manner through the present process, generating virtually no wastewater and eliminating the need for subsequent wastewater treatment, resulting in excellent economic and environmental benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of natural active ingredient extraction, and in particular to a process for extracting and purifying chlorogenic acid from honeysuckle and purified chlorogenic acid extracted by the process, and particularly to the application of the purified chlorogenic acid in the technical field of food safety. Background Art

[0002] Foodborne pathogens are a major food safety risk and a major cause of foodborne illness. Common antimicrobial ingredients include inorganic, synthetic, and natural antimicrobial ingredients. Metal ions with antimicrobial activity are typical examples of inorganic antimicrobial ingredients. While metal ions offer advantages such as high antimicrobial efficiency and a broad antimicrobial spectrum, research has shown that metal ions and metal-based nanoparticles are commonly associated with cytotoxicity. Synthetic organic antimicrobial ingredients, such as phenols, biguanides, and halamine compounds, have been widely studied. While these antimicrobial ingredients exhibit broad-spectrum and high-efficiency antimicrobial activity, they often suffer from inherent toxicity or the potential for decomposition to produce toxic substances. Natural antimicrobial ingredients, with their diverse sources and diverse types, offer promising application prospects. Currently, chitosan, plant essential oils, antimicrobial peptides, lysozymes, bacteriophages, bacteriocins, organic acids, flavonoids, and polyphenols are among those most studied both domestically and internationally.

[0003] Honeysuckle, a plant of the genus Lonicera in the family Caprifoliaceae, is widely distributed in Henan, Shandong, and other regions of my country. As an important traditional Chinese medicine, honeysuckle boasts heat-clearing and detoxifying properties, as well as anti-inflammatory and swelling-reducing properties. It is a key ingredient in traditional Chinese medicines such as Lianhua Qingwen Capsules, Yinqiao Jiedu Tablets, Shuanghuanglian Oral Liquid, and Yinhuang Granules. The primary active ingredient in honeysuckle is chlorogenic acid, an organic acid with antibacterial, antiviral, anti-tumor, anti-aging, antioxidant, free radical scavenging, and blood pressure-lowering and lipid-lowering properties. As a natural antimicrobial agent, chlorogenic acid has significant potential for application in food safety.

[0004] At present, the main methods for extracting chlorogenic acid from honeysuckle include water extraction, ethanol extraction, ultrasound or microwave-assisted extraction, enzymatic hydrolysis, etc., and the main methods for purifying chlorogenic acid include macroporous resin method, membrane separation method, ethyl acetate method, gel column chromatography, high-speed countercurrent chromatography, etc. However, many current extraction and purification methods have problems such as low purity and environmental pollution. At the same time, the extraction consumes a lot of supplies and generates a lot of waste, which is not conducive to green and sustainable development. For example, the ethanol extraction method requires a large amount of organic solvents and requires subsequent treatment; the macroporous resin method requires regular regeneration of the resin, and the regeneration process requires the use of acids, alkalis, organic solvents, etc., which is time-consuming and the waste generated requires subsequent treatment. Therefore, it is of great significance to develop environmentally friendly and sustainable chlorogenic acid extraction and purification processes. Summary of the Invention

[0005] In view of this, on the one hand, in order to solve the technical problems of low purity, environmental pollution and large amount of waste generated in the extraction and purification methods in the prior art, the present invention provides a process for extracting and purifying chlorogenic acid from honeysuckle. The purity of the purified chlorogenic acid finally prepared can reach more than 90%. It only uses three reagents: water, ethanol, and ethyl acetate. The ethanol and ethyl acetate used can be closed-loop reused through the process of the present invention, and almost no waste liquid is generated. There is no need for subsequent waste liquid treatment, which produces good economic and environmental benefits.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A process for extracting and purifying chlorogenic acid from honeysuckle comprises the following steps:

[0008] Step (1), grinding honeysuckle leaves or branches into powder to obtain honeysuckle powder;

[0009] Step (2), extracting the honeysuckle powder in step (1) with water;

[0010] Step (3), after the water extraction is completed, filtering is performed to preliminarily remove insoluble impurities to obtain honeysuckle precipitate and supernatant, and the supernatant is further removed of insoluble impurities to obtain a clarified supernatant;

[0011] Step (4), drying the clarified supernatant obtained in step (2) to preliminarily obtain a purified crude chlorogenic acid powder;

[0012] Step (5), taking the purified crude chlorogenic acid powder obtained in step (4), adding an acidic aqueous solution to prepare a crude chlorogenic acid aqueous solution;

[0013] Step (6), adding ethyl acetate to the crude chlorogenic acid aqueous solution obtained in step (5), shaking and extracting, so that the chlorogenic acid migrates from water to ethyl acetate, to obtain a mixed solution 1;

[0014] Step (7), separating the mixed solution obtained in step (6) and allowing it to stand, after the upper ethyl acetate layer and the lower water layer are completely separated, taking the upper ethyl acetate, adding an alkaline aqueous solution to the ethyl acetate, and extracting by shaking to transfer the chlorogenic acid from the ethyl acetate to the water, thereby obtaining a mixed solution II;

[0015] Step (8), separating the mixed solution 2 in step (7) and letting it stand, after the upper ethyl acetate layer and the lower chlorogenic acid aqueous solution are completely separated, taking the lower chlorogenic acid aqueous solution and drying it to obtain purified chlorogenic acid powder;

[0016] Step (9), taking the purified chlorogenic acid powder obtained in step (8), adding pure water to prepare a chlorogenic acid aqueous solution, adding ethyl acetate to the chlorogenic acid aqueous solution, shaking extraction, so that the chlorogenic acid migrates from the water to the ethyl acetate, and drying to obtain further purified chlorogenic acid powder;

[0017] Step (10), taking the further purified chlorogenic acid powder obtained in step (9), adding an ethanol-ethyl acetate mixed reagent, performing oscillation extraction and suction filtration to obtain a filtrate and a filter residue;

[0018] Step (11): The filtrate obtained in step (10) is evaporated, concentrated and crystallized. When a large amount of suspended matter is observed, the evaporation is stopped, the filter is filtered and the filter residue is dried to obtain the final purified chlorogenic acid powder.

[0019] Preferably, after step (8) and before step (9), the method further includes step (81):

[0020] Repeat steps (6)-(8).

[0021] Preferably, after step (9) and before step (10), the method further includes step (91):

[0022] Repeat steps (7)-(81).

[0023] Preferably, the alkaline aqueous solution in step (7) is replaced with pure water.

[0024] Preferably, the method further comprises repeating step (5).

[0025] Preferably, the method further includes step (12), repeating step (11).

[0026] Preferably, the method further comprises step (13), wherein the filter residue obtained in step (10) is repeatedly extracted and crystallized, and the obtained final purified chlorogenic acid powder is combined.

[0027] Preferably, the method further comprises step (14), combining the filtrate residues obtained in steps (11) to (13), repeating the crystallization, and combining the obtained final purified chlorogenic acid powder.

[0028] Preferably, the ethyl acetate in step (6) is new analytically pure ethyl acetate;

[0029] The ethyl acetate used after step (6) is the ethyl acetate used previously.

[0030] On the other hand, the present invention provides a purified chlorogenic acid, which is prepared according to the above process for extracting and purifying chlorogenic acid from honeysuckle, and has a purity of more than 90%.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a process for extracting and purifying chlorogenic acid from honeysuckle, and the purity of the purified chlorogenic acid finally prepared can reach more than 90%. Only three reagents, water, ethanol, and ethyl acetate, are used. The ethanol and ethyl acetate used can be reused in a closed loop through the process of the present invention, and almost no waste liquid is generated. No subsequent waste liquid treatment is required, resulting in good economic and environmental benefits.

[0033] Other beneficial effects of the present invention will be described in detail in the following specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a process flow chart of the present invention;

[0035] Figure 2 This is the HPLC spectrum of the aqueous extract of honeysuckle branches and leaves;

[0036] Figure 3 is the HPLC standard curve of chlorogenic acid; DETAILED DESCRIPTION

[0037] like Figure 1 As shown, the present invention provides a process for extracting and purifying chlorogenic acid from honeysuckle, comprising the following steps:

[0038] Step (1), grinding the honeysuckle leaves or branches into powder to obtain honeysuckle powder, wherein the honeysuckle powder preferably has a particle size of 40-200 mesh;

[0039] Step (2), adding water to the honeysuckle powder in step (1), heating and stirring, the ratio of honeysuckle powder to water is preferably 1:8 to 1:40 (by weight), more preferably 1:15 to 1:20, the temperature is preferably 30 to 100 degrees, more preferably 60 to 80 degrees, and the extraction time is preferably 15 minutes to 3 hours, more preferably 45 minutes to 1 hour;

[0040] After the water extraction in step (3) and step (2) is completed, the mixture is filtered through a 50-300 mesh filter to preliminarily remove insoluble impurities to obtain a honeysuckle precipitate and a supernatant, and the supernatant is further subjected to the removal of insoluble impurities (preferably by the following method) to obtain a clarified supernatant;

[0041] The following three methods were used to further remove insoluble impurities from the supernatant:

[0042] Method 1: Use 1 micron or 5 micron PP cotton for further filtration, but the consumption of PP cotton is large and it is not renewable;

[0043] Method 2: Add 1% PAC flocculant to the supernatant slowly, stirring and observing the changes in the solution. When obvious particles appear, stop adding and stirring, and let it stand to obtain a clear supernatant.

[0044] Method 3: Centrifuge the supernatant at a speed of 4500-12000 rpm, for 3-5 minutes, and 1-3 times to obtain a clarified supernatant.

[0045] Methods 2 and 3 are more preferred, wherein in method 3, the preferred rotation speed is 10,000-12,000 rpm and the number of centrifugation is 2-3 times. The honeysuckle precipitate is further extracted with water, and the extraction is repeated 2-4 times, and the resulting supernatant is combined with the supernatant of the first extraction;

[0046] Step (4), drying the clarified supernatant obtained in step (2) by a spray dryer to preliminarily obtain a purified crude chlorogenic acid powder;

[0047] The content of the purified crude chlorogenic acid powder is preferably detected by high performance liquid chromatography. The spray dryer parameters are preferably set to: fan 100%, temperature 160-220 degrees, more preferably 160-180 degrees, peristaltic pump 20-60%, more preferably 30-50%.

[0048] Step (5): Take the purified crude chlorogenic acid powder obtained in step (4), add an acidic aqueous solution, and prepare a crude chlorogenic acid aqueous solution. The ratio of the purified crude chlorogenic acid powder to the acidic aqueous solution is preferably 1:4-1:11 (by weight), more preferably 1:5-1:8, and the pH of the acidic aqueous solution is preferably 0-5, more preferably 0-2. The acidic aqueous solution is prepared from hydrochloric acid and pure water.

[0049] Step (6), adding ethyl acetate to the crude chlorogenic acid aqueous solution obtained in step (5), shaking and extracting, so that the chlorogenic acid migrates from the water to the ethyl acetate, and obtaining a mixed solution (which may be referred to as a chlorogenic acid aqueous solution). The ratio of the crude chlorogenic acid aqueous solution to ethyl acetate is preferably 1:1 to 1:5 (by volume), more preferably 1:1.5 to 1:2.5 (by volume), the extraction time is preferably 10-60 minutes, more preferably 10-15 minutes, the shaking speed is preferably 50-300 rpm, more preferably 150-200 rpm, and the extraction temperature is preferably 10-70 degrees, more preferably 15-35 degrees;

[0050] After the step (7) and the extraction process is completed, the mixed solution obtained in step (6) is transferred to a separatory funnel and allowed to stand until the upper ethyl acetate layer and the lower aqueous layer are completely separated. At this time, the aqueous layer contains a certain amount of chlorogenic acid. The upper ethyl acetate is taken, and an alkaline aqueous solution is added to the ethyl acetate, and the chlorogenic acid is extracted by oscillation to migrate from the ethyl acetate to the water to obtain a mixed solution 2 (chlorogenic acid alkaline aqueous solution). The pH of the alkaline aqueous solution is preferably 8-13, more preferably 10-12, the alkaline aqueous solution is prepared from sodium hydroxide and pure water, the ratio of the ethyl acetate extract to the alkaline aqueous solution is preferably 5:1 to 1:1 (by volume), more preferably 2:1 to 1:1 (by volume), the extraction time is preferably 10-60 minutes, more preferably 10-15 minutes, the oscillation speed is preferably 50-300 rpm, more preferably 150-200 rpm, and the extraction temperature is preferably 10-70 degrees, more preferably 15-35 degrees.

[0051] The principles of steps (6)-(7) above are as follows:

[0052] Chlorogenic acid is an organic acid. Under acidic conditions, the dissociation of chlorogenic acid is restricted, and it mainly exists in molecular form, tending to migrate from water to ethyl acetate. Under alkaline conditions, chlorogenic acid reacts with alkali to become an ionic form (acid radical), tending to migrate from ethyl acetate to water.

[0053] Step (8), after the above extraction process is completed, the mixed solution in step (7) is transferred to a separating funnel and allowed to stand. After the water layer (lower layer) and the ethyl acetate layer (upper layer) are completely separated, the lower layer of chlorogenic acid aqueous solution is taken and dried by a spray dryer to obtain purified chlorogenic acid powder, and its content is detected by high performance liquid chromatography;

[0054] In order to further improve the recovery rate of chlorogenic acid, the present invention further includes step (81):

[0055] Repeat steps (6)-(8), specifically:

[0056] The water layer after step (6) still contains a large amount of chlorogenic acid. According to the parameters of step (6), ethyl acetate is added to the crude chlorogenic acid aqueous solution that has been extracted once for two extractions to obtain an ethyl acetate solution containing chlorogenic acid. According to the parameters of step (7), an alkaline aqueous solution is added to the ethyl acetate solution containing chlorogenic acid for extraction to obtain an aqueous solution containing chlorogenic acid. According to step (8), spray drying is performed to obtain further purified chlorogenic acid powder, which is combined with the purified chlorogenic acid powder obtained in step (8) and regarded as the same batch of products.

[0057] Among them, except for the first extraction using new analytically pure ethyl acetate, the subsequent extraction uses the previously used ethyl acetate (the ethyl acetate used in the first oscillation extraction in step (6)), thereby forming a closed process, which can maximize the avoidance or reduction of the generation of ethyl acetate waste liquid.

[0058] The experimental results show that the use of previously used ethyl acetate does not adversely affect the purity of the final chlorogenic acid product (as illustrated by the following examples and comparative examples).

[0059] The aqueous solution after multiple extractions in step (6) still contains chlorogenic acid. Preferably, referring to step (5), crude chlorogenic acid powder is further added thereto (the purified crude chlorogenic acid powder obtained in step (4) is taken, and an acidic aqueous solution is added to prepare a crude chlorogenic acid aqueous solution; the aqueous solution after multiple extractions in step (6) still contains chlorogenic acid (at this time, it is still an acidic aqueous solution containing chlorogenic acid), and the chlorogenic acid therein cannot be wasted. However, since the chlorogenic acid content therein is relatively low after multiple extractions, the purified crude chlorogenic acid powder obtained in step (4) is added thereto to increase the chlorogenic acid concentration in the acidic aqueous solution, so as to facilitate subsequent more efficient repeated extractions). This forms a closed process, which can avoid or reduce the generation of acidic waste liquid to the greatest extent possible, and at the same time, can minimize the loss of chlorogenic acid.

[0060] Existing extraction methods leave chlorogenic acid in the aqueous solution after extraction, essentially wasting it. Furthermore, since the solution is acidic, wastewater treatment is required, which is environmentally unfriendly and increases costs. Although some existing methods have shown that chlorogenic acid is largely depleted in aqueous solution after three extractions, our hundreds of experimental data suggest that these reports may not be true. Therefore, we fully utilize the remaining chlorogenic acid-containing aqueous solution by re-adding crude chlorogenic acid powder to it, creating a dynamic, sustainable, and uninterrupted extraction process that avoids the generation of wastewater and the waste of chlorogenic acid.

[0061] The experimental results show that the continued addition of chlorogenic acid powder to the aqueous solution after multiple extractions does not adversely affect the purity of the final chlorogenic acid product (as illustrated by the following examples and comparative examples).

[0062] Step (9): Take the purified chlorogenic acid powder obtained in step (8), add pure water to prepare a chlorogenic acid aqueous solution, add ethyl acetate to the chlorogenic acid aqueous solution, shake and extract, so that the chlorogenic acid migrates from the water to the ethyl acetate, and dry to obtain further purified chlorogenic acid powder. The ratio of the purified chlorogenic acid powder to water is preferably 1:4-1:8 (by weight), more preferably 1:5-1:6, the ratio of the chlorogenic acid aqueous solution to ethyl acetate is preferably 1:1 to 1:5 (by volume), more preferably 1:1.5 to 1:2.5 (by volume), the extraction time is preferably 10-30 minutes, more preferably 10-15 minutes, the shaking speed is preferably 100-200 rpm, more preferably 150-200 rpm, and the extraction temperature is preferably 10-50 degrees, more preferably 15-35 degrees.

[0063] To further improve the recovery rate, the present invention also includes step (91), which is specifically:

[0064] Repeat the above steps (7)-(81), except that the alkaline aqueous solution in step (7) is replaced with pure water. Drying in a spray dryer can obtain further purified chlorogenic acid powder, and its content is detected by high performance liquid chromatography;

[0065] Step (10): Take the further purified chlorogenic acid powder obtained in step (9), add an ethanol-ethyl acetate mixed reagent, perform oscillation extraction and suction filtration to obtain a filtrate and a filter residue. The mixing ratio of ethanol and ethyl acetate is preferably 1:1 to 1:9 (by volume), more preferably 1:2 to 1:4 (by volume), the oscillation speed is preferably 50-300 rpm, more preferably 150-200 rpm, the extraction time is preferably 5-60 minutes, more preferably 5-15 minutes, and the extraction temperature is preferably 10-80 degrees, more preferably 20-40 degrees.

[0066] In this step, we preferably add an ethanol-ethyl acetate mixed reagent. Although a methanol-ethyl acetate mixed reagent can also be used, we have found through a large number of experiments that the use of ethanol is more effective. Moreover, ethanol is safer to use. The toxicity of methanol is relatively high. Conventionally used in the prior art is a methanol-ethyl acetate mixed system.

[0067] Step (11): The filtrate obtained in step (10) is evaporated, concentrated, and crystallized. When a large amount of suspended matter is observed, the evaporation is stopped, and the filter is filtered to obtain a filter residue, which is dried to obtain the final purified chlorogenic acid powder. The temperature selected for evaporation and crystallization is preferably 20-100 degrees, more preferably 60-80 degrees, and the selected equipment is preferably a rotary evaporator and a fume hood.

[0068] The present invention further includes step (12), repeating step (11) 1-4 times, and preferably repeating the evaporation crystallization 2-3 times.

[0069] The present invention further includes step (13), wherein the filter residue obtained in step (10) is repeatedly extracted, preferably 2-6 times, and crystallized repeatedly, and the resulting purified chlorogenic acid powder is combined and regarded as the same batch of products. The filter residue remaining from the last extraction is incorporated into chlorogenic acids 1-4 for recycling, thereby minimizing the loss of chlorogenic acid. Experimental results show that incorporating the filter residue containing chlorogenic acid into unextracted chlorogenic acid powder does not adversely affect the purity of the final chlorogenic acid product (as illustrated by the following examples and comparative examples).

[0070] The present invention further includes step (14), combining the filtrate residues obtained in steps (11) to (13), repeating the crystallization, and combining the obtained final purified chlorogenic acid powders, which are regarded as the same batch of products.

[0071] On the other hand, the present invention provides a purified chlorogenic acid, which is prepared according to the above process for extracting and purifying chlorogenic acid from honeysuckle, and has a purity of more than 90%.

[0072] The technical solution of the present invention is described in detail below with reference to specific embodiments.

[0073] like Figure 2 As shown, the HPLC spectrum of the aqueous extract of honeysuckle branches and leaves

[0074] Take 2 grams of honeysuckle branches and leaves respectively, grind them into powder, add 28 grams of water, 37 degrees, 200rpm, and shake and extract for 30 minutes. Filter with a 300-mesh filter, centrifuge the filtrate at a speed of 10,000 rpm for 3 minutes, obtain the supernatant, and measure HPLC. The HPLC parameters are as follows: (1) The mobile phase is 0.5% phosphoric acid aqueous solution and methanol, and the ratio of the two is: 25% methanol and 75% 0.5% phosphoric acid aqueous solution from 0 to 16 minutes; after 16 minutes, it is 55% methanol and 45% 0.5% phosphoric acid aqueous solution; (2) The injection volume is 10 microliters; (3) The flow rate is 0.8 mL / min; (4) The detection wavelength is 351 nm; (5) The HPLC column is a C18 column, 5um, 4.6×150 mm. According to the above parameter conditions, the attached Figure 2 .

[0075] like Figure 3 As shown, the HPLC standard curve of chlorogenic acid was drawn

[0076] Take 20 mg of chlorogenic acid standard and add 10 g of pure water to prepare a 2 mg / mL chlorogenic acid standard aqueous solution. Dilute with pure water to prepare standard solutions with concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0 mg / mL, respectively. HPLC measurements (column specifications: C18, 5-Micron, 4.6 × 150 mm; instrument model: Agilent HPLC 1290) were performed. A standard curve was drawn based on the peak height and peak area values. The relationship between chlorogenic acid concentration and peak height was y = 1815.5x, and the relationship between chlorogenic acid concentration and peak area was y = 17351x.

[0077] Example 1

[0078] Extraction of purified crude chlorogenic acid

[0079] Take 4730 grams of honeysuckle leaves, grind them into powder, add 50 kg of water, stir and heat to 80 degrees, extract for 1 hour, cool to room temperature, filter, centrifuge at 4500 rpm for 5 minutes, and spray dry (blower 100%, temperature 180 degrees, peristaltic pump 40%). Extract 3 times to obtain 1048 grams of purified crude chlorogenic acid with a yield of 22%. HPLC analysis showed that the content was 3.3% based on the peak height. Repeat this step to collect about 10 kilograms of purified crude chlorogenic acid, which is convenient for subsequent processes.

[0080] The purified crude chlorogenic acid was purified by acidic aqueous solution-ethyl acetate-alkaline aqueous solution

[0081] 200 g of purified crude chlorogenic acid was ground into powder, 1000 g of an acidic aqueous solution with a pH of 1 was added, and the mixture was stirred to dissolve. 2000 g of ethyl acetate was then added, and the mixture was extracted at 15 degrees and 200 rpm for 10 minutes. The mixture was allowed to stand for stratification. The upper ethyl acetate layer was taken, and 1400 g of a sodium hydroxide aqueous solution with a pH of 11 was added. The mixture was extracted at 15 degrees and 200 rpm for 10 minutes. The mixture was allowed to stand for stratification, and the lower chlorogenic acid alkaline aqueous solution was collected. 1700 g of ethyl acetate was added to the acidic chlorogenic acid aqueous solution after ethyl acetate extraction, and the mixture was extracted at 15 degrees and 200 rpm for 10 minutes. The mixture was allowed to stand for stratification, and the lower chlorogenic acid alkaline aqueous solution was collected. The lower chlorogenic acid alkaline aqueous solution was combined and spray-dried to obtain 4.14 g of chlorogenic acid powder. HPLC analysis showed that the content was 47.8% based on the peak height. Repeat this step and finally collect about 100 grams of purified chlorogenic acid powder to facilitate subsequent processes.

[0082] Chlorogenic acid was further purified by water-ethyl acetate-water

[0083] 18 g of chlorogenic acid powder purified with ethyl acetate was ground into powder, added to 180 g of pure water, and stirred to dissolve. 900 g of ethyl acetate was then added, and the mixture was extracted with shaking at 20°C and 300 rpm for 10 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 900 g of ethyl acetate was added. The mixture was extracted with shaking at 20°C and 300 rpm for 10 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 1100 g of ethyl acetate was added. The mixture was extracted with shaking at 20°C and 300 rpm for 10 minutes. The upper ethyl acetate layer after three extractions was pooled, 600 g of water was added, and the mixture was extracted with shaking at 20°C and 300 rpm for 10 minutes. The upper ethyl acetate layer was removed, and 600 g of water was added. The mixture was extracted with shaking at 20°C and 300 rpm for 10 minutes. The lower chlorogenic acid aqueous solution was combined and spray-dried to obtain 0.77 g of further purified chlorogenic acid powder. HPLC analysis showed that the content was 67.0% based on the peak height. Repeat this step and finally collect about 10 grams of further purified chlorogenic acid powder to facilitate the subsequent process.

[0084] Chlorogenic acid was crystallized by evaporation of ethanol-ethyl acetate mixed solvent

[0085] Ethanol and ethyl acetate were mixed in a volume ratio of 1 to 3 to prepare a mixed solvent. 2.5 g of chlorogenic acid powder further purified by water-ethyl acetate-water was added to 5000 ml of the mixed solvent and extracted at 20 degrees and 300 rpm for 10 minutes. The mixture was filtered and the filtrate was added to a rotary evaporator and concentrated at 40 degrees. After a large amount of white suspended matter appeared, the mixed solution was filtered, the filter residue was collected, and the chlorogenic acid powder was washed and dried. 5000 ml of the mixed solvent was added to the chlorogenic acid filter residue extracted once, and the above steps were repeated. The collected chlorogenic acid powders were combined. The chlorogenic acid powder after evaporation and crystallization was taken and the above steps were repeated for two and three recrystallizations. The chlorogenic acid powder collected after the three recrystallizations was measured by HPLC, and its content was calculated based on the peak height to be 91.2%.

[0086] Example 2

[0087] The use of previously used ethyl acetate will not adversely affect the purity of the final chlorogenic acid product, as follows:

[0088] The purified crude chlorogenic acid was purified by acidic aqueous solution-used ethyl acetate-alkaline aqueous solution

[0089] 100 g of the crude chlorogenic acid purified in Example 1 was ground into a powder and added to 800 g of an acidic aqueous solution at pH 2. The mixture was stirred and dissolved. 1600 g of used ethyl acetate was then added and extracted with shaking at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for separation. The upper ethyl acetate layer was removed and 1100 g of a sodium hydroxide aqueous solution at pH 12 was added. The mixture was shaken and extracted at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for separation. The lower alkaline chlorogenic acid aqueous solution was collected. 1200 g of used ethyl acetate was added to the acidic chlorogenic acid aqueous solution extracted with used ethyl acetate. The mixture was shaken and extracted at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for separation. The upper ethyl acetate layer was removed and 1000 g of a sodium hydroxide aqueous solution at pH 12 was added. The mixture was shaken and extracted at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for separation. The lower alkaline chlorogenic acid aqueous solution was collected. The lower alkaline chlorogenic acid aqueous solution was combined and spray-dried to obtain 2.56 g of chlorogenic acid powder. HPLC analysis showed that the content of the chlorogenic acid powder was 48.2% based on the peak height. Repeat this step and finally collect about 50 grams of purified chlorogenic acid powder to facilitate the subsequent process.

[0090] Chlorogenic acid was further purified by water-used ethyl acetate-water

[0091] 13 g of chlorogenic acid powder purified with used ethyl acetate was ground into a powder, added to 130 g of pure water, and stirred to dissolve. 650 g of used ethyl acetate was then added, and the mixture was extracted with shaking at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 520 g of ethyl acetate was added. The mixture was extracted with shaking at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 780 g of ethyl acetate was added. The mixture was extracted with shaking at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for stratification. The upper ethyl acetate layer after three extractions was pooled, 500 g of water was added, and the mixture was extracted with shaking at 20°C and 200 rpm for 12 minutes. The upper ethyl acetate layer was removed, and 500 g of water was added. The mixture was extracted with shaking at 20°C and 200 rpm for 12 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was combined and spray-dried to obtain 0.84 g of further purified chlorogenic acid powder. HPLC analysis showed a content of 68.7% based on peak height.

[0092] Chlorogenic acid was crystallized by evaporation of a mixed solvent of ethanol and used ethyl acetate

[0093] Ethanol and used ethyl acetate were mixed in a volume ratio of 1 to 3 to prepare a mixed solvent. 0.6 g of chlorogenic acid powder, further purified by water-used ethyl acetate-water, was added to 1200 ml of the mixed solvent and extracted at 20°C, 200 rpm, and shaken for 12 minutes. The filtrate was filtered and placed on a rotary evaporator and concentrated at 65°C. After a large amount of white suspended matter appeared, the mixed solution was filtered, the filter residue was collected, washed, and dried to obtain chlorogenic acid powder. An additional 1000 ml of the mixed solvent was added to the chlorogenic acid residue from the first extraction, and the above steps were repeated. The collected chlorogenic acid powders were combined. The chlorogenic acid powder after evaporation and crystallization was taken and the above steps were repeated for a second and third recrystallization. The chlorogenic acid powder collected after the third recrystallization was analyzed by HPLC, and the content was calculated based on the peak height to be 92.8%.

[0094] Comparative Example 2

[0095] The purified crude chlorogenic acid was purified by acidic aqueous solution-fresh ethyl acetate-alkaline aqueous solution

[0096] The same method as in Example 2 was used, except that the used ethyl acetate in Example 2 was replaced with fresh, unused ethyl acetate. The resulting product was 2.12 g of chlorogenic acid powder. HPLC analysis revealed a 47.3% content based on peak height. This procedure was repeated, ultimately yielding approximately 50 g of purified chlorogenic acid powder to facilitate subsequent processing.

[0097] Chlorogenic acid was further purified by water-fresh ethyl acetate-water

[0098] The same method as Example 2 was used, except that the used ethyl acetate in Example 2 was replaced with fresh, unused ethyl acetate. The remaining steps were the same. 0.84 g of further purified chlorogenic acid powder was obtained. HPLC analysis revealed a content of 67.4% based on peak height.

[0099] Chlorogenic acid was evaporated and crystallized by ethanol-new ethyl acetate mixed solvent

[0100] The same method as Example 2 was used except that the used ethyl acetate in Example 2 was replaced with fresh, unused ethyl acetate. The chlorogenic acid powder collected after three recrystallizations was analyzed by HPLC, and the content of the chlorogenic acid was calculated based on the peak height to be 92.1%.

[0101] Chlorogenic acid content in Example 2 and Comparative Example 2

[0102]

[0103]

[0104] It can be seen from the contents of the above table that the use of previously used ethyl acetate does not have an adverse effect on the purity of the final chlorogenic acid product.

[0105] Example 3

[0106] Continuing to add chlorogenic acid powder to the aqueous solution after multiple extractions will not adversely affect the purity of the final chlorogenic acid product

[0107] Take the chlorogenic acid aqueous solution extracted 3 times and continue to add chlorogenic acid powder

[0108] 800 g of the chlorogenic acid aqueous solution extracted three times was added to 100 g of the crude chlorogenic acid purified in Example 1, stirred and dissolved, and then 2000 g of ethyl acetate was added. The mixture was shaken and extracted at 20 degrees and 200 rpm for 15 minutes. The mixture was allowed to stand for stratification. The upper layer of ethyl acetate was taken, and 1000 g of a sodium hydroxide aqueous solution with a pH of 12 was added. The mixture was shaken and extracted at 20 degrees and 200 rpm for 15 minutes. The mixture was allowed to stand for stratification, and the lower layer of the chlorogenic acid alkaline aqueous solution was collected. 1500 g of ethyl acetate was further added to the acidic aqueous solution of chlorogenic acid after ethyl acetate extraction. The mixture was shaken and extracted at 20 degrees and 200 rpm for 15 minutes. The mixture was allowed to stand for stratification. The upper layer of ethyl acetate was taken, and 1000 g of a sodium hydroxide aqueous solution with a pH of 12 was added. The mixture was shaken and extracted at 20 degrees and 200 rpm for 15 minutes. The mixture was allowed to stand for stratification, and the lower layer of the chlorogenic acid alkaline aqueous solution was collected. Continue to add 1000 g of ethyl acetate to the acidic aqueous solution of chlorogenic acid after ethyl acetate extraction, shake and extract at 20 degrees and 200 rpm for 15 minutes, let it stand and separate, take the upper layer of ethyl acetate, add 1000 g of sodium hydroxide aqueous solution with pH = 12, shake and extract at 20 degrees and 200 rpm for 15 minutes, let it stand and separate, and collect the lower layer of chlorogenic acid alkaline aqueous solution. A total of 3 extractions. The extracted lower layer of chlorogenic acid alkaline aqueous solution was combined and spray-dried to obtain 3.85 g of chlorogenic acid powder. HPLC analysis showed that the content was 46.8% based on the peak height. Repeat this step, and finally collect about 50 g of purified chlorogenic acid powder to facilitate the subsequent process.

[0109] Chlorogenic acid was further purified by water-ethyl acetate-water

[0110] 15 g of the chlorogenic acid powder purified with ethyl acetate was ground into powder, added to 160 g of pure water, and stirred to dissolve. 720 g of used ethyl acetate was then added, and the mixture was extracted with shaking at 30°C and 150 rpm for 15 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 580 g of ethyl acetate was added. The mixture was extracted with shaking at 30°C and 150 rpm for 15 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was removed, and 830 g of ethyl acetate was added. The mixture was extracted with shaking at 30°C and 150 rpm for 15 minutes. The mixture was allowed to stand for stratification. The upper ethyl acetate layer after three extractions was pooled, 600 g of water was added, and the mixture was extracted with shaking at 30°C and 150 rpm for 15 minutes. The mixture was allowed to stand for stratification. The upper ethyl acetate layer was removed, and 600 g of water was added. The mixture was extracted with shaking at 30°C and 150 rpm for 15 minutes. The mixture was allowed to stand for stratification. The lower chlorogenic acid aqueous solution was combined and spray-dried to obtain 1.22 g of further purified chlorogenic acid powder. HPLC analysis showed a content of 69.4% based on peak height.

[0111] Chlorogenic acid was crystallized by evaporation of ethanol-ethyl acetate mixed solvent

[0112] Ethanol and ethyl acetate were mixed in a volume ratio of 1 to 4 to prepare a mixed solvent. 0.9 g of chlorogenic acid powder further purified by water-used ethyl acetate-water was added to 1600 ml of the mixed solvent and extracted at 15 degrees and 200 rpm for 15 minutes. The mixture was filtered and the filtrate was added to a rotary evaporator and concentrated at 70 degrees. After a large amount of white suspended matter appeared, the mixed solution was filtered, the filter residue was collected, washed and dried to obtain chlorogenic acid powder. 1200 ml of the mixed solvent was added to the chlorogenic acid filter residue extracted once, and the above steps were repeated. The collected chlorogenic acid powders were combined. The chlorogenic acid powder after evaporation and crystallization was taken and the above steps were repeated for two and three recrystallizations. The chlorogenic acid powder collected after the three recrystallizations was measured by HPLC, and its content was calculated based on the peak height to be 93.5%.

[0113] Comparative Example 3

[0114] The purified crude chlorogenic acid was purified by acidic aqueous solution-ethyl acetate-alkaline aqueous solution

[0115] The procedure in Example 3 was modified from "800 g of chlorogenic acid aqueous solution extracted three times and added to 100 g of purified crude chlorogenic acid" to "100 g of purified crude chlorogenic acid was ground into a powder and added to 800 g of an acidic aqueous solution at pH 2." The remaining steps remained the same. This yielded 3.24 g of chlorogenic acid powder. HPLC analysis revealed a 46.4% content based on peak height. This step was repeated, ultimately yielding approximately 50 g of purified chlorogenic acid powder, which facilitated subsequent processing.

[0116] Chlorogenic acid was further purified by water-ethyl acetate-water

[0117] The same method as Example 3 was used, except that different chlorogenic acid powder was used. 1.15 g of further purified chlorogenic acid powder was obtained. HPLC analysis revealed a content of 67.2% based on peak height.

[0118] Chlorogenic acid was crystallized by evaporation of ethanol-ethyl acetate mixed solvent

[0119] Same as Example 3. The only difference was that different chlorogenic acid powder was used. HPLC analysis showed that the content of chlorogenic acid was 93.0% based on the peak height.

[0120] Chlorogenic acid content in Example 3 and Comparative Example 3

[0121]

[0122] Example 4

[0123] Incorporating the chlorogenic acid-containing filter residue into the unextracted chlorogenic acid powder does not adversely affect the purity of the final chlorogenic acid product;

[0124] Extraction of purified crude chlorogenic acid

[0125] Same as Example 1.

[0126] The purified crude chlorogenic acid was purified by acidic aqueous solution-ethyl acetate-alkaline aqueous solution

[0127] Same as Example 1.

[0128] Chlorogenic acid was further purified by water-ethyl acetate-water

[0129] Same as Example 1.

[0130] Chlorogenic acid was crystallized by evaporation of ethanol-ethyl acetate mixed solvent

[0131] Ethanol and ethyl acetate were mixed in a volume ratio of 1 to 2.5 to prepare a mixed solvent. 1.9 g of the filter residue containing chlorogenic acid extracted twice with ethanol and ethyl acetate in Example 1 was added to 2.1 g of the chlorogenic acid powder obtained by further purification in this example (Example 4) by water, ethyl acetate, and water, for a total of 4.0 g. 3500 ml of the mixed solvent was added, and the mixture was shaken and extracted at 40 degrees and 150 rpm for 15 minutes. The mixture was filtered and the filtrate was added to a rotary evaporator and concentrated at 60 degrees. After a large amount of white suspended matter appeared, the mixed solution was filtered, the filter residue was taken, and the chlorogenic acid powder was washed and dried. HPLC analysis showed that the content was 94.7% based on the peak height.

[0132] Comparative Example 4

[0133] Same as Example 4. The only difference is that "1.9 g of the filter residue containing chlorogenic acid extracted twice with ethanol and ethyl acetate in Example 1 was added with 2.1 g of unextracted chlorogenic acid powder, for a total of 4.0 g." is replaced with "4.0 g of the chlorogenic acid powder obtained by further purification with water, ethyl acetate, and water in this Example (Example 4)." HPLC analysis showed a content of 94.5% based on peak height.

[0134] Chlorogenic acid content in Example 4 and Comparative Example 4

[0135]

[0136] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present invention shall be covered by the scope of protection of the present invention.

Claims

1. A process for extracting and purifying chlorogenic acid from honeysuckle, characterized in that: The steps include: Step (1), grinding honeysuckle leaves or branches into powder to obtain honeysuckle powder; Step (2), extracting the honeysuckle powder in step (1) with water; Step (3), after the water extraction is completed, filtering is performed to preliminarily remove insoluble impurities to obtain honeysuckle precipitate and supernatant, and the supernatant is further removed of insoluble impurities to obtain a clarified supernatant; Step (4), drying the clarified supernatant obtained in step (2) to preliminarily obtain a purified crude chlorogenic acid powder; Step (5), taking the purified crude chlorogenic acid powder obtained in step (4), adding an acidic aqueous solution to prepare a crude chlorogenic acid aqueous solution; Step (6), adding ethyl acetate to the crude chlorogenic acid aqueous solution obtained in step (5), shaking and extracting, so that the chlorogenic acid migrates from water to ethyl acetate, to obtain a mixed solution 1; Step (7), separating the mixed solution obtained in step (6) and allowing it to stand, after the upper ethyl acetate layer and the lower water layer are completely separated, taking the upper ethyl acetate, adding an alkaline aqueous solution to the ethyl acetate, and extracting by shaking to transfer the chlorogenic acid from the ethyl acetate to the water, thereby obtaining a mixed solution II; Step (8), separating the mixed solution 2 in step (7) and letting it stand, after the upper ethyl acetate layer and the lower chlorogenic acid aqueous solution are completely separated, taking the lower chlorogenic acid aqueous solution and drying it to obtain purified chlorogenic acid powder; Step (9), taking the purified chlorogenic acid powder obtained in step (8), adding pure water to prepare a chlorogenic acid aqueous solution, adding ethyl acetate to the chlorogenic acid aqueous solution, shaking extraction, so that the chlorogenic acid migrates from the water to the ethyl acetate, and drying to obtain further purified chlorogenic acid powder; Step (10), taking the further purified chlorogenic acid powder obtained in step (9), adding an ethanol-ethyl acetate mixed reagent, performing oscillation extraction and suction filtration to obtain a filtrate and a filter residue; Step (11): The filtrate obtained in step (10) is evaporated, concentrated and crystallized. When a large amount of suspended matter is observed, the evaporation is stopped, the filter is filtered and the filter residue is dried to obtain the final purified chlorogenic acid powder.

2. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 1, characterized in that: After step (8) and before step (9), the method further includes step (81): Repeat steps (6)-(8).

3. A process for extracting and purifying chlorogenic acid from honeysuckle according to claim 2, characterized in that: After step (9) and before step (10), the method further includes step (91): Repeat steps (7)-(81).

4. A process for extracting and purifying chlorogenic acid from honeysuckle according to claim 3, characterized in that: The alkaline aqueous solution in step (7) was replaced with pure water.

5. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 1, characterized in that: It also includes repeating step (5).

6. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 1, characterized in that: The method further includes step (12), repeating step (11).

7. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 6, characterized in that: The method further comprises step (13), wherein the filter residue obtained in step (10) is repeatedly extracted and crystallized, and the obtained final purified chlorogenic acid powder is combined.

8. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 7, characterized in that: The method further comprises step (14), combining the filtrate residues obtained in steps (11) to (13), repeating the crystallization, and combining the obtained final purified chlorogenic acid powder.

9. The process for extracting and purifying chlorogenic acid from honeysuckle according to claim 1, characterized in that: The ethyl acetate in step (6) is new analytically pure ethyl acetate; The ethyl acetate used after step (6) is the ethyl acetate used previously.

Images