Method for extracting and separating chlorogenic acid from honeysuckle flower

Abstract

The invention belongs to the technical field of extraction processes, and particularly relates to a method for extracting and separating chlorogenic acid from flos lonicerae. The method comprises the following steps: a, carrying out reflux extraction, filtration and concentration on flos lonicerae, leaves of the flos lonicerae or a flower and leaf mixture of the flos lonicerae to obtain extract, and centrifuging to obtain centrifugate; b, adjusting the pH value of the centrifugate to be acidic, removing impurities through macroporous adsorption resin 1, and eluting with a weak base solution to obtain an alkaline water eluent; c, adjusting the pH value of the alkaline water eluent to be acidic, removing impurities through macroporous adsorption resin 2, and eluting with an ethanol solution to obtain an alcohol eluent; and d, concentrating the alcohol eluent into extract, refrigerating, filtering and drying to obtain a chlorogenic acid crude product, adding a solvent, dissolving, refrigerating, filtering and drying to obtain the chlorogenic acid. According to the method, the yield and the purity of the extracted chlorogenic acid are improved; the method is simple and feasible, the production cost is reduced, the adopted reagent is environment-friendly, the obtained product is better in safety, and industrial production is easy.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction technology, and particularly relates to a method for extracting and separating chlorogenic acid from honeysuckle. Background Art

[0002] Chlorogenic acid (CGA) is a very important natural phenolic acid compound, widely found in plants. It exhibits a variety of biological activities, including antioxidant, anti-inflammatory, hypoglycemic, and anti-tumor properties. It is widely used in medicine, food, and cosmetics. Chlorogenic acid is primarily extracted from plants, with green coffee beans, Eucommia leaves, burdock leaves, and honeysuckle being the main sources of chlorogenic acid both domestically and internationally.

[0003] The 2010 edition of the Sichuan Provincial Standard for Traditional Chinese Medicines describes Sichuan honeysuckle as the dried buds or early blooming flowers of Lonicera similis Hemsl. and Lonicera acuminata Wall., both species of the Caprifoliaceae family. The former is commonly known as "Nanjiang Silver Flower," while the latter is known as "Belly Silver Flower." The drafting instructions for the Sichuan Provincial Standard for Traditional Chinese Medicines indicate that the average chlorogenic acid content in 21 batches of Sichuan honeysuckle (two basal species) from different locations was 6.6%, with a maximum of 11.3%. Our measurements show that the chlorogenic acid content in Sichuan honeysuckle leaves also exceeds 4%. Therefore, the isolation and extraction of high-purity chlorogenic acid from Sichuan honeysuckle and its leaves holds great promise.

[0004] Existing chlorogenic acid extraction processes are complex, costly, and involve the use of numerous toxic organic reagents. Patent application number CN200510000484.0 discloses a process for extracting chlorogenic acid from burdock leaves. This patented technology utilizes a large amount of organic solvents such as chloroform and ethyl acetate, and the process steps are complex. Long-term exposure to toxic organic solvents not only poses a serious health hazard to operators but also causes persistent pollution and irreversible damage to the surrounding ecological environment. Therefore, developing an efficient and environmentally friendly method for extracting and isolating high-concentration chlorogenic acid from honeysuckle is particularly important. Summary of the Invention

[0005] The object of the present invention is to provide a method for extracting and separating chlorogenic acid from Lonicera japonica, which solves the technical problems of low yield, low purity and environmental pollution of chlorogenic acid extraction in the prior art.

[0006] The present invention provides a method for extracting and separating chlorogenic acid from Lonicera japonica, comprising the following steps:

[0007] a. Reflux extract the honeysuckle, its leaves, or its flowers and leaves, filter, concentrate to an extract, and centrifuge to obtain a centrifuge;

[0008] b. Adjust the pH value of the centrifuge liquid to acidic, pass it through macroporous adsorption resin 1 to remove impurities, and elute it with a weak alkaline solution to obtain an alkaline eluate;

[0009] c. Adjust the pH value of the alkaline eluate to acidic, pass it through macroporous adsorption resin 2 to remove impurities, and elute it with ethanol solution to obtain an alcohol eluate;

[0010] d. Concentrate the alcohol eluate to an extract, refrigerate, filter, and dry to obtain crude chlorogenic acid, add a solvent to dissolve, refrigerate, filter, and dry to obtain the extract.

[0011] Furthermore, in step a, the solvent used in the reflux extraction is an ethanol solution with a volume concentration of 30-70%; the added amount is 8-10 times the total weight of the raw materials.

[0012] Preferably, the solvent is an ethanol solution with a volume concentration of 50%.

[0013] Furthermore, in step a, the reflux extraction is performed 1 to 3 times, each time for 1 to 2 hours.

[0014] Furthermore, in step a, the concentration is reduced-pressure concentration; and the temperature of the reduced-pressure concentration is 50-90°C.

[0015] Preferably, the temperature for concentration under reduced pressure is 50°C.

[0016] Furthermore, in step a, the concentration of the extract is 0.1 g crude drug / mL to 0.5 g crude drug / mL.

[0017] Furthermore, in step b, the pH value of the centrifuge is adjusted to 2-5.

[0018] Preferably, the pH value of the centrifuge is adjusted to 3.

[0019] The model of the macroporous adsorption resin 1 is HPD-100, HPD-400, AB-8 or ADS-7.

[0020] Preferably, the model of the macroporous adsorption resin 1 is HPD-100 or HPD-400.

[0021] The loading amount of the centrifuge solution passing through the macroporous adsorption resin 1 is 0.1 g to 0.5 g of crude drug per 1 mL of column volume.

[0022] Preferably, the loading amount is 0.2 g to 0.4 g of crude drug per 1 mL column volume.

[0023] Most preferably, the loading amount is 0.2 g of crude drug per 1 mL column volume.

[0024] Furthermore, the sample loading flow rate of the macroporous adsorption resin 1 is 1.0 to 4.0 times the column volume.

[0025] Preferably, the sample loading flow rate of the macroporous adsorption resin 1 is 2.0 to 3.0 times the column volume.

[0026] Furthermore, in step b, the weak base is any one of sodium carbonate, disodium hydrogen phosphate, ammonia water or sodium acetate.

[0027] Preferably, the weak alkaline solution is a disodium hydrogen phosphate solution having a concentration of 0.2% to 0.4%.

[0028] Preferably, the concentration of the disodium hydrogen phosphate solution is 0.4%.

[0029] Furthermore, in step c, the pH value of the alkaline water eluent is adjusted to 2-5.

[0030] Preferably, the pH value of the alkaline water eluent is adjusted to 3.

[0031] Furthermore, in step c, the model of the macroporous adsorption resin 2 is HPD-100, HPD-400, AB-8 or ADS-7.

[0032] Preferably, the model of the macroporous adsorption resin 2 is HPD-100 or HPD-400.

[0033] Furthermore, in step c, the loading amount of the centrifuge liquid passing through the macroporous adsorption resin 2 is 0.1 g to 0.5 g of crude drug per 1 mL of column volume.

[0034] Preferably, the loading amount is 0.2 g to 0.4 g of crude drug per 1 mL column volume.

[0035] Most preferably, the loading amount is 0.2 g of crude drug per 1 mL column volume.

[0036] Furthermore, in step c, the loading flow rate of the macroporous adsorption resin 2 is 1.0 to 4.0 times the column volume.

[0037] Preferably, the sample loading flow rate of the macroporous adsorption resin 2 is 2.0 to 3.0 times the column volume.

[0038] Furthermore, in step c, the volume concentration of the ethanol solution is 20% to 60%.

[0039] Preferably, the volume concentration of the ethanol solution is 30%.

[0040] Furthermore, in step d, the alcohol eluate is concentrated to a concentration of the extract of 2.0 g crude drug / mL to 5.0 g crude drug / mL.

[0041] Furthermore, in step d, the refrigeration time is 1 to 5 days.

[0042] Furthermore, in step d, the drying temperature is 50-80°C.

[0043] Furthermore, in step d, the solvent is any one of water, 30% volume concentration methanol solution, 50% volume concentration methanol solution, 70% volume concentration methanol solution or methanol.

[0044] Preferably, the solvent is water.

[0045] The beneficial effects of the present invention are:

[0046] The present invention prepares a centrifuge from honeysuckle by passing it through a macroporous resin column 1, removing impurities, eluting with a weak alkaline solution, and then passing it through a macroporous adsorption resin column 2 for purification. This improves the yield and purity of chlorogenic acid. The yield of crude chlorogenic acid can reach 75.4% and the purity can reach 98%, while the yield of refined chlorogenic acid can reach 60.2% and the purity can reach 99.8%. The method of the present invention is simple and feasible, reduces production costs, and the reagents used in the present invention are environmentally friendly. The resulting product has better safety and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a diagram showing the results of investigating the amount of eluent used in Example 7.

[0048] Figure 2 This is a graph showing the results of investigating the amount of 0.4% disodium hydrogen phosphate solution used for elution in Example 7. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below through specific examples. The drugs and reagents involved in the specific embodiments are all commercially available.

[0050] Example 1

[0051] Weigh 400 g of honeysuckle (chlorogenic acid content: 8.62%), add 50% ethanol solution by volume, and reflux extract three times, each time for 1 hour, adding 4000 mL for the first time, and 3200 mL for the second and third times. Filter, combine the filtrates, and concentrate under reduced pressure at 50°C to 2000 ml, and centrifuge to obtain a centrifuge;

[0052] The pH value of the centrifuge was adjusted to 3.0 with a hydrochloric acid solution, and the centrifuge was passed through an HPD-100 macroporous adsorption resin column 1 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 12.0 L of a 0.4% disodium hydrogen phosphate solution, and the alkaline eluate was collected;

[0053] The pH value of the alkaline eluate was adjusted to 3.0, and the eluate was passed through an HPD-100 macroporous adsorption resin column 2 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 6.0 L of a 30% ethanol solution by volume, and the alcohol eluate was collected;

[0054] The alcohol eluate was concentrated under reduced pressure at 50°C to 200 mL, refrigerated for 1 day, filtered, and dried at 50°C to obtain crude chlorogenic acid. The crude product was dissolved in water, refrigerated for 1 day, filtered, and dried at 50°C to obtain the product.

[0055] Crude chlorogenic acid yield: 75.3%, purity: 98.0%;

[0056] The yield of refined chlorogenic acid is 60.2% and the purity is 99.8%.

[0057] Example 2

[0058] Weigh 400 g of honeysuckle (chlorogenic acid content: 8.62%), add 50% ethanol solution by volume, and reflux extract 3 times, each time for 1 hour, adding 4000 mL for the first time, and 3200 mL for the second and third times. Filter, combine the filtrates, and concentrate under reduced pressure at 50°C to 1000 mL, and centrifuge to obtain a centrifuge;

[0059] The pH value of the centrifuge was adjusted to 3.0 with a hydrochloric acid solution, and the centrifuge was passed through an HPD-400 macroporous adsorption resin column 1 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 12.0 L of a 0.4% disodium hydrogen phosphate solution, and the alkaline eluate was collected;

[0060] The pH value of the alkaline eluate was adjusted to 3.0, and the eluate was passed through an HPD-100 macroporous adsorption resin column 2 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 6.0 L of a 30% ethanol solution by volume, and the alcohol eluate was collected;

[0061] The alcohol eluate was concentrated under reduced pressure at 50°C to 200 mL, refrigerated for 5 days, filtered, and dried at 50°C to obtain crude chlorogenic acid. The crude product was dissolved in water, refrigerated for 5 days, filtered, and dried at 50°C to obtain the crude product.

[0062] The yield of crude chlorogenic acid is 74.8%, and the purity is 97.8%.

[0063] The yield of refined chlorogenic acid is 59.5%, and the purity is 99.6%.

[0064] Example 3

[0065] Weigh 400 g of mixed medicinal materials of Sichuan honeysuckle flowers and leaves (chlorogenic acid content: 5.65%), add 50% ethanol solution by volume, and reflux extract 3 times, each time for 1 hour, adding 4000 mL for the first time, and 3200 mL for the second and third times. Filter, combine the filtrates, and concentrate under reduced pressure at 50°C to 1000 mL, and centrifuge to obtain a centrifuge;

[0066] The pH of the centrifuge solution was adjusted to 3.0 with a hydrochloric acid solution, and the centrifuge solution was passed through an HPD-400 macroporous adsorption resin column 1 with a column volume of 2000 mL and a sample loading flow rate of 3.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 12.0 L of a 0.4% disodium hydrogen phosphate solution, and the alkaline eluate was collected;

[0067] The pH value of the alkaline eluate was adjusted to 3.0, and the eluate was passed through an HPD-400 macroporous adsorption resin column 2 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 6.0 L of a 30% ethanol solution by volume, and the alcohol eluate was collected;

[0068] The alcohol eluate was concentrated under reduced pressure at 50°C to 100 mL, refrigerated for 3 days, filtered, and dried at 50°C to obtain crude chlorogenic acid. The crude product was dissolved in water, refrigerated for 3 days, filtered, and dried at 50°C to obtain crude chlorogenic acid.

[0069] Crude chlorogenic acid yield: 68.6%, purity: 97.7%;

[0070] The yield of refined chlorogenic acid is 54.2%, and the purity is 99.2%.

[0071] Example 4

[0072] Weigh 400 g of mixed medicinal materials of flowers and leaves of Sichuan honeysuckle (chlorogenic acid content: 5.65%), add 50% ethanol solution by volume, and reflux extract 3 times, each time for 1 hour, adding 4000 mL for the first time, and 3200 mL for the second and third times. Filter, combine the filtrates, and concentrate under reduced pressure at 50°C to 800 mL, and centrifuge to obtain a centrifuge;

[0073] The pH value of the centrifuge was adjusted to 3.0 with a hydrochloric acid solution, and the centrifuge was passed through an HPD-100 macroporous adsorption resin column 1 with a column volume of 2000 mL and a sample loading flow rate of 3.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 12.0 L of a 0.4% disodium hydrogen phosphate solution, and the alkaline eluate was collected;

[0074] The pH value of the alkaline eluate was adjusted to 3.0, and the eluate was passed through an HPD-400 macroporous adsorption resin column 2 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 6.0 L of a 30% ethanol solution by volume, and the alcohol eluate was collected;

[0075] The alcohol eluate was concentrated under reduced pressure at 50°C to 130 mL, refrigerated for 2 days, filtered, and dried at 50°C to obtain crude chlorogenic acid. The crude product was dissolved in water, refrigerated for 2 days, filtered, and dried at 50°C to obtain crude chlorogenic acid.

[0076] The yield of crude chlorogenic acid is 65.4%, and the purity is 96.6%.

[0077] The yield of refined chlorogenic acid is 50.2%, and the purity is 98.7%.

[0078] Example 5

[0079] Weigh 400 g of Lonicera japonica leaves (chlorogenic acid content: 4.5%), add 50% ethanol solution by volume, and reflux extract three times for 1 hour each time, adding 4000 mL for the first time, and 3200 mL for the second and third times. Filter, combine the filtrates, and concentrate under reduced pressure at 50°C to 1330 mL. Centrifuge to obtain a centrifuge.

[0080] The pH value of the centrifuge was adjusted to 3.0 with a hydrochloric acid solution, and the centrifuge was passed through an HPD-100 macroporous adsorption resin column 1 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 12.0 L of a 0.4% disodium hydrogen phosphate solution, and the alkaline eluate was collected;

[0081] The pH value of the alkaline eluate was adjusted to 3.0, and the eluate was passed through an HPD-100 macroporous adsorption resin column 2 with a column volume of 2000 mL and a sample loading flow rate of 2.0 BV / h. Impurities were removed with 12.0 L of purified water with a pH value of 3.0, and then eluted with 6.0 L of a 30% ethanol solution by volume, and the alcohol eluate was collected;

[0082] The alcohol eluate was concentrated under reduced pressure at 50°C to 80 mL, refrigerated for 4 days, filtered, and dried at 50°C to obtain crude chlorogenic acid. The crude product was dissolved in water, refrigerated for 4 days, filtered, and dried at 50°C to obtain crude chlorogenic acid.

[0083] Crude chlorogenic acid yield: 62.2%, purity: 92.9%;

[0084] The yield of refined chlorogenic acid is 46.6%, and the purity is 96.2%.

[0085] Example 6 Investigation of extraction process conditions

[0086] 6.1 Investigation of Extraction Solvents for Lonicera japonica

[0087] Ten portions of Flos Lonicerae (50 g each) were collected, divided into two groups, and extracted three times with ethanol solutions of 30%, 40%, 50%, 60%, and 70% by volume, for one hour each. 500 mL was added for the first extraction, and 400 mL for the second and third extractions. The extracts were filtered, the filtrates combined, and cooled. The chlorogenic acid content in the different extraction solvents was determined. The results are shown in Table 1.

[0088] Table 1 Results of investigation of extraction solvents (n=2)

[0089] Ethanol solution concentration 30％ 40％ 50％ 60％ 70％ Extraction amount (g) 3.52 4.04 4.21 4.20 4.16 Extraction rate (%) 81.67 93.73 97.68 97.45 96.52

[0090] The results showed that the extraction rates of 50% ethanol and 60% ethanol were higher. Considering the production cost, 50% ethanol was selected as the extraction solvent.

[0091] 6.2 Test on absorption of extraction solvent by medicinal materials

[0092] Take 12 portions of Sichuan honeysuckle, 50 g / portion, and group two. Add 400 mL of 50% ethanol solution to each group. After 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, filter, weigh the medicinal materials, and calculate the results. See Table 2.

[0093] Table 2 Percentage survey results of medicinal materials (n=2)

[0094] Soaking time (h) 0.5 1.0 1.5 2.0 2.5 3.0 Absorption rate (%) 192 195 201 199 203 202

[0095] The experimental results show that the absorption rate of the extraction solvent by the medicinal materials is about 200%, which is twice the amount of the medicinal materials.

[0096] 6.3 Extraction orthogonal test

[0097] There are many factors that affect the extraction effect of chlorogenic acid in Sichuan honeysuckle. Through the analysis of the physical and chemical properties of the substances contained, it was decided to use the number of extractions, extraction time, and the amount of 50% ethanol solution added as the investigation factors. Combined with production practice, three levels were designed for each factor. The experimental plan is shown in Table 3.

[0098] Table 3 Factor level table

[0099]

[0100] According to the factor level table, select L9(3 4) was used to conduct the experiment. This table can accommodate up to four three-level factors. This experiment only had three factors, which were randomly arranged in the table, leaving the fourth column blank. The experimental factors and levels were assigned to the corresponding columns, and the experimental results were entered in Table 4. Chlorogenic acid extraction was used as the evaluation indicator, and the experimental results were analyzed by variance analysis.

[0101] Table 4 Orthogonal test results (n=2)

[0102]

[0103] The results of the ANOVA test showed that the critical F values ​​for the F test were F0.05(2,2)=19.0 and F0.01(2,2)=99.0. The detailed results are shown in Table 5.

[0104] Table 5 Results of variance analysis

[0105] Sources of variance Sum of Squares of Deviations degrees of freedom F-number Significance Number of extractions A 5.987 2 145.72 ** Extraction time B 0.044 2 1.078 Material-liquid ratio C 0.447 2 10.875 Error e 0.041 2

[0106] The test results show that the difference in factor A (number of extractions) is significant, so A3 is selected; the difference between factors B and C is not significant; combined with the results of range analysis, and considering production efficiency and production cost savings, B1 and C2 are selected.

[0107] Based on the above analysis, the optimal extraction process is determined to be: A3B1C2. At the same time, combined with the test results of the medicinal materials absorption of extraction solvents, 2BV more solvent is added during the first extraction. Therefore, the extraction process is: add 50% ethanol and reflux extraction three times, adding 10BV for the first time, and 8BV for the second and third times, and each extraction is 1 hour.

[0108] 6.4 Extraction process validation

[0109] Experiments were conducted based on the optimal process conditions determined by orthogonal experiments to investigate the yield of chlorogenic acid.

[0110] Two portions of honeysuckle (400 g each) were weighed and extracted three times with 50% ethanol (1 hour each). The first extraction was performed with 4000 mL of ethanol, followed by 3200 mL of ethanol for the second and third extractions. The extracts were filtered, the combined filtrates cooled, and the chlorogenic acid content of the extracts was determined. The results are shown in Table 6.

[0111] Table 6 Extraction process verification results (n=2)

[0112] Chlorogenic acid content (g) Chlorogenic acid extraction rate (%) 34.00 98.61

[0113] The test results show that after adding 50% volume concentration of ethanol solution and reflux extraction three times, adding 10BV for the first time, and adding 8BV for the second and third times, each extraction for 1 hour, the amount of chlorogenic acid is 34.00g, and the extraction rate of chlorogenic acid is 98.61%.

[0114] Example 7 Screening of Resin Purification Process Route

[0115] Preparation of centrifuge for purification process: weigh 400 g / portion of Sichuan honeysuckle, add 50% ethanol solution by volume and extract three times, each time for 1 hour; add 4000 mL for the first time, and 3200 mL for the second and third times, filter, combine the filtrate, recover ethanol under reduced pressure at 50°C and concentrate to 2000 mL, centrifuge, take the centrifuge, add appropriate amount of water to make a solution of 0.2 g crude drug / mL, and obtain.

[0116] 7.1 Investigation of resin models

[0117] Eight centrifuges (250 mL each, equivalent to 50 g of medicinal material) were measured and adjusted to pH 3.0. The mixture was then passed through pre-treated HPD-100, HPD-400, AB-8, and ADS-7 macroporous adsorption resin columns (400 mL column volume). The columns were rinsed with 2400 mL of purified water, and the effluent was collected. Elution was then performed with 1200 mL of 30% ethanol, and the eluate was collected. The flow rate was 2 BV / h. The total amount of chlorogenic acid in the eluates from the different resin types was determined. The results are shown in Table 7.

[0118] Table 7 Results of resin model investigation (n=2)

[0119] Resin Model HPD-100 HPD-400 AB-8 ADS-7 Total amount of effluent (g) 0.11 0.15 0.83 0.03 Total amount of eluent (g) 4.13 4.09 3.43 1.97

[0120] The test results showed that ADS-7 had the best adsorption effect of chlorogenic acid, followed by HPD-100 and HPD-400; however, ADS-7 had the worst elution effect, and there was no significant difference in the elution effects of HPD-100 and HPD-400, so the resin models selected were HPD-100 and HPD-400.

[0121] 7.2 Determination of pH value of drug solution before loading

[0122] Eight aliquots of the centrifuge solution (250 mL each, equivalent to 50 g of medicinal material) were measured and adjusted to pH 2.0, 3.0, 4.0, and 5.0, respectively. The solution was then passed through an HPD-100 macroporous adsorption resin column (400 mL column volume). The column was rinsed with 2400 mL of purified water at the corresponding pH value. The eluate was then eluted with 1200 mL of 30% ethanol solution, and the eluate was collected at a flow rate of 2 BV / h. The total amount of chlorogenic acid and total solids in the eluate were determined. The results are shown in Table 8.

[0123] Table 8 Results of pH value investigation of pre-column drug solution (n=2)

[0124] pH 2.0 3.0 4.0 5.0 Total chlorogenic acid (g) 4.12 4.11 3.54 1..25 Total solids (g) 7.01 7.08 6.90 4.45

[0125] The test results show that the total amount of chlorogenic acid and total solid content are the highest when the pH value is 2.0-3.0, but less acid is used when the pH value is 3.0, so the pH value of the drug solution before column loading is selected as 3.0.

[0126] 7.3 Investigation of drug solution loading amount

[0127] 200 mL, 400 mL, 600 mL, 800 mL, and 1000 mL of the centrifuge solution (equivalent to 40 g, 80 g, 120 g, 160 g, and 200 g of the crude drug, respectively) were measured and adjusted to pH 3.0. The solution was then passed through an HPD-100 macroporous adsorption resin column (column volume, 400 mL). The column was rinsed with 2400 mL of pH 3.0 purified water. Elution was then performed with 1200 mL of 30% ethanol solution, and the eluate was collected at a flow rate of 2 BV / h. The total amount of chlorogenic acid in the eluate was determined. The results are shown in Table 9.

[0128] Table 9 Results of investigation on the amount of drug solution loaded (n=2)

[0129] Sample amount (total amount of crude drug) 40g 80g 120g 160g 200g Total chlorogenic acid (g) 3.33 6.53 6.60 6.77 6.83

[0130] The experimental results show that when the sample load on the 400 mL column volume is 80 g of crude drug (i.e., 0.2 g of crude drug per 1 mL column volume), the adsorption amount of chlorogenic acid has reached saturation. In order to ensure the adsorption effect of chlorogenic acid in the drug solution, 0.2 g of crude drug per 1 mL column volume is selected.

[0131] 7.4 Investigation of the flow rate of drug solution loading

[0132] Eight portions of the centrifuge solution (200 mL / portion, equivalent to 40 g of crude drug) were measured and adjusted to pH 3.0. The solution was then passed through an HPD-100 macroporous adsorption resin column (column volume, 200 mL) at flow rates of 1, 2, 3, and 4 BV / h, respectively. The column was rinsed with 1200 mL of pH 3.0 purified water and then eluted with 600 mL of 30% ethanol solution. Both the rinse and elution flow rates were 2 BV / h. The total amount of chlorogenic acid in the eluate was determined. The results are shown in Table 10.

[0133] Table 10 Results of investigation on the flow rate of drug solution loading

[0134] Loading flow rate (BV / h) 1 2 3 4 Total chlorogenic acid (g) 3.28 2.29 3.28 3.01

[0135] The experimental results show that the total amount of chlorogenic acid is slightly affected when the sample flow rate is 1BV / h, 2BV / h, and 3BV / h, but the total amount of chlorogenic acid decreases significantly when it reaches 4BV / h. Therefore, a sample flow rate of 2BV / h to 3BV / h is more reasonable.

[0136] 7.5 Selection of eluent

[0137] Eight portions of the centrifuge solution (200 mL / portion, equivalent to 40 g of crude drug) were measured and adjusted to pH 3.0. The solution was passed through an HPD-100 macroporous adsorption resin chromatography column (column volume, 200 mL). The column was rinsed with 1200 mL of purified water (pH 3.0), and then eluted with 600 mL of 20% ethanol, 30% ethanol, 40% ethanol, and 50% ethanol solutions, respectively, at a flow rate of 2 BV / h. The eluates were collected. The total amount of chlorogenic acid in the eluates was determined. The results are shown in Table 11.

[0138] Table 11 Eluent investigation results (n=2)

[0139] Elution concentration (%) 20 30 40 50 Total chlorogenic acid (g) 2.35 3.29 3.28 3.30

[0140] The test results showed that when the ethanol concentration reached 30% or more, there was no significant difference in the total amount of chlorogenic acid in the eluent, so an ethanol solution with a volume concentration of 30% was selected as the eluent.

[0141] 7.6 Determination of eluent dosage

[0142] Measure 2 portions of the centrifuge liquid, 200 mL portions (equivalent to 40 g of crude drug), adjust the pH to 3.0, pass through an HPD-100 macroporous adsorption resin chromatography column (column volume 200 mL), rinse the column with 1200 ml of pH 3.0 purified water, and then elute with a 30% volume concentration of ethanol solution at a flow rate of 2 BV / h. Use 0.5 times the column bed volume as each point for segmented collection, and collect eluates of 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 times the column bed volume. By detecting the total amount of chlorogenic acid in each segment of the eluate, draw an elution curve to determine the collection amount and dosage of the eluent. The results of the investigation are shown in Table 12 and Figure 1 .

[0143] Table 12 Results of investigation on eluent dosage (n=2)

[0144] Eluent (times) 0.50 1.00 1.50 2.00 2.50 3.00 Total chlorogenic acid (g) 2.19 0.95 0.10 0.03 0.01 0.01

[0145] The test results showed that the elution rate of chlorogenic acid reached 99.4% when the amount of eluent collected reached 2 column volumes. Therefore, the total amount of eluent collected was determined to be 2 to 3 column volumes, and the amount of eluent used was selected to be 3 column volumes.

[0146] 7.7 Determination of Eluent Flow Rate

[0147] Eight portions of the centrifuge (200 mL / portion, equivalent to 40 g of crude drug) were measured and the pH was adjusted to 3.0. The mixture was passed through an HPD-100 macroporous adsorption resin chromatography column (column volume, 200 mL). The column was rinsed with 1200 mL of pH 3.0 purified water at a flow rate of 2.0 BV / h. The column was then eluted with 600 mL of 30% ethanol solution at flow rates of 1.0 BV / h, 2 BV / h, 3 BV / h, and 4 BV / h, respectively. The eluate was collected. The total amount of chlorogenic acid and total solids in the eluate were determined. The results are shown in Table 13.

[0148] Table 13 Eluent flow rate investigation results (n=2)

[0149] Elution flow rate (times) 1.0 2.0 3.0 4.0 Total chlorogenic acid (g) 3.28 3.28 3.29 3.29 Total solids (g) 5.66 5.68 5.69 5.68 Chlorogenic acid / total solids (%) 58.26 58.36 58.13 58.33

[0150] The test results show that the eluent flow rate of 1.0-4.0 BV / h has no significant effect on the total amount of chlorogenic acid and the total solid content, and the amount of chlorogenic acid accounts for 58% of the total solid content, that is, the purity of the sample prepared by this process can reach 58%.

[0151] 7.8 Screening of base types

[0152] Eight portions of the centrifuge (200 mL / portion, equivalent to 40 g of crude drug) were measured and adjusted to pH 3.0. The mixture was passed through an HPD-100 macroporous adsorption resin chromatography column (column volume, 200 mL). The column was rinsed with 1200 mL of pH 3.0 purified water and eluted with 1200 mL each of 0.4% sodium carbonate solution, 0.4% disodium hydrogen phosphate solution, 0.4% aqueous ammonia solution, and 0.4% sodium acetate solution, all at a flow rate of 2.0 BV / h. The eluate was collected. The total amount of chlorogenic acid in the eluate was determined. The results are shown in Table 14.

[0153] Table 14 Results of investigation on alkali species (n=2)

[0154] Types of alkali sodium carbonate Disodium hydrogen phosphate ammonia Sodium acetate Total chlorogenic acid (g) 3.25 3.27 3.26 2.93

[0155] The test results show that sodium carbonate, disodium hydrogen phosphate and ammonia water all have good elution effects; at the same time, sodium carbonate will produce bubbles during elution, affecting the elution efficiency; ammonia water has a pungent odor, so disodium hydrogen phosphate was chosen.

[0156] 7.9 Investigation of Disodium Hydrogen Phosphate Concentration

[0157] Six portions of the centrifuge solution (200 mL / portion, equivalent to 40 g of crude drug) were measured and adjusted to pH 3.0. The solution was passed through an HPD-100 macroporous adsorption resin chromatography column (column volume, 200 mL). The column was rinsed with 1200 mL of pH 3.0 purified water and eluted with 1200 mL each of 0.2% disodium hydrogen phosphate solution, 0.3% disodium hydrogen phosphate solution, and 0.4% disodium hydrogen phosphate solution at a flow rate of 2.0 BV / h. The eluates were collected. The total amount of chlorogenic acid in the eluates was determined. The results are shown in Table 15.

[0158] Table 15 Alkali concentration investigation results (n=2)

[0159] concentration 0.20％ 0.30％ 0.40％ Total chlorogenic acid (g) 2.29 2.98 3.27

[0160] The test results show that the elution effect is best when the elution concentration of disodium hydrogen phosphate is 0.4%.

[0161] 7.10 Study on the amount of 0.4% disodium hydrogen phosphate solution used for elution

[0162] Measure 2 portions of the centrifuge liquid, 200 mL / portion (equivalent to 40 g of crude drug), adjust the pH to 3.0, pass through an HPD-100 macroporous adsorption resin chromatography column (column volume 200 mL), rinse the column with 1200 mL of pH 3.0 purified water, and then elute with 0.4% disodium hydrogen phosphate solution at a flow rate of 2 BV / h. Use 1.0 times the column bed volume as each point for segmented collection, and collect 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 times the column bed volume of eluate. By detecting the total amount of chlorogenic acid in each segment of the eluate, draw an elution curve to determine the collection amount and dosage of the eluent. The results of the investigation are shown in Table 16 and Figure 2 .

[0163] Table 16 Results of investigation on eluent dosage (n=2)

[0164] Eluent (times) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Total chlorogenic acid (g) 1.69 0.81 0.47 0.22 0.07 0.03 0.01

[0165] The test results show that the amount of chlorogenic acid in the eluate is already low when the amount of eluate collected reaches 5 times the column volume. Considering the comprehensive production efficiency, the total amount of eluate collected is determined to be 5 to 6 times the column volume, that is, the amount of eluent used is 6 times the column volume.

[0166] 7.11 Determination of Eluent Flow Rate

[0167] Eight portions of the centrifuge (200 ml each, equivalent to 40 g of crude drug) were measured and adjusted to pH 3.0. The mixture was passed through an HPD-100 macroporous adsorption resin chromatography column (column volume, 200 ml). The column was rinsed with 1200 ml of pH 3.0 purified water at a flow rate of 2.0 BV / h. Elution was then performed with 1200 ml of 0.4% disodium hydrogen phosphate solution at flow rates of 1.0 BV / h, 2 BV / h, 3 BV / h, and 4 BV / h, respectively. The eluate was collected. The total amount of chlorogenic acid and total solids in the eluate were determined. The results are shown in Table 17.

[0168] Table 17 Eluent flow rate investigation results (n=2)

[0169] Elution flow rate (times) 1.0 2.0 3.0 4.0 Total chlorogenic acid (g) 3.29 3.28 3.25 3.19 Total solids (g) 5.33 5.32 5.32 5.3 Chlorogenic acid / total solids (%) 61.73 61.65 61.09 60.19

[0170] The experimental results show that the eluent flow rate of 1.0-3.0 BV / h has no significant effect on the total amount of chlorogenic acid and the total solid content, and the amount of chlorogenic acid accounts for more than 61% of the total solid content, that is, the purity of the sample prepared by this process can reach 61%.

[0171] 7.12 Investigation of Purification Process Route

[0172] Take 2 portions of the centrifuge liquid, 200 mL / portion (equivalent to 40 g of crude drug), adjust the pH value to 3.0, pass through HPD-100 macroporous adsorption resin chromatography column 1 (column volume is 200 mL), rinse the column with 1200 mL of pH 3.0 purified water, and then elute with 1200 mL of 0.4% disodium hydrogen phosphate solution at a flow rate of 2 BV / h, collect the eluate, adjust the pH value of the eluate to 3.0, pass through HPD-100 macroporous adsorption resin chromatography column 2 (column volume is 200 mL), rinse the column with 1200 mL of pH 3.0 purified water, and then elute with 600 mL of 4% sodium hydrogen phosphate solution. Elution was performed with 30% ethanol at a flow rate of 2 BV / h. The eluate was collected and the total amount of chlorogenic acid and total solids in the eluate was determined. The results showed that the total amount of chlorogenic acid was 3.19 g, the total solids was 3.71 g, and the total amount of chlorogenic acid / total solids was 85.98%. That is, the purity of the sample prepared by this process can reach 85.98%. See Table 18

[0173] Table 18 Purification process route screening results

[0174]

[0175] The results showed that the preparation process of route three had a transfer rate of chlorogenic acid of 93.82% and a purity of 85.98%, and the process route was feasible.

[0176] 7.13 Selection of Crystallization Solvent

[0177] Weigh 400 g of Lonicera japonica. Prepare a 30% ethanol eluate according to purification route three. Recover the ethanol under reduced pressure at 50°C and concentrate until the eluate is alcohol-free. Dry at 50°C and pulverize to obtain 37.5 g of the extract. Weigh 10 portions (1.87 g / portion) and add appropriate amounts of water, 30% methanol, 50% methanol, 70% methanol, and methanol, respectively. Dissolve the extract by ultrasonication. Add the corresponding solvent to 10 mL, mix well, and refrigerate for 3 days. Remove the extract, filter, collect the filter cake, and dry at 50°C to obtain the crude chlorogenic acid extract. The extract was weighed and the chlorogenic acid content was determined. The results are shown in Table 19.

[0178] Table 19 Crystallization solvent selection results

[0179] solvent water 30% methanol 50% methanol 70% methanol Methanol Total crude extract (g) 1.34 1.12 1.01 0.86 0.80 Chlorogenic acid content (%) 98.02 95.23 92.50 92.41 92.00

[0180] The results show that water has the highest yield and total amount of crystallization and is more environmentally friendly. At the same time, using water can simplify the production process and reduce the concentration and drying steps, so water is selected as the crystallization solvent.

Claims

1. A method for extracting and isolating chlorogenic acid from Lonicera japonica, characterized by: The steps include: a. Reflux extract the honeysuckle flower, its leaves, or its flowers and leaves, filter, concentrate to an extract, and centrifuge to obtain a centrifuge; b. Adjust the pH value of the centrifuge liquid to acidic, pass it through macroporous adsorption resin 1 to remove impurities, and elute it with a weak alkaline solution to obtain an alkaline eluate; c. Adjust the pH value of the alkaline eluate to acidic, pass it through macroporous adsorption resin 2 to remove impurities, and elute it with ethanol solution to obtain an alcohol eluate; d. Concentrate the alcohol eluate to an extract, refrigerate, filter, and dry to obtain crude chlorogenic acid, add a solvent to dissolve, refrigerate, filter, and dry to obtain the extract.

2. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step a, the solvent used in the reflux extraction is an ethanol solution with a volume concentration of 30-70%; the added amount is 8-10 times the total weight of the raw materials.

3. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1 or 2, characterized in that: In step a, the reflux extraction is performed 1 to 3 times, each time for 1 to 2 hours.

4. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, wherein: In step a, the concentration of the extract is 0.1 g crude drug / mL to 0.5 g crude drug / mL.

5. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step b, at least one of the following is met: Adjust the pH value of the centrifuge to 2-5; The model of the macroporous adsorption resin 1 is HPD-100, HPD-400, AB-8 or ADS-7; The loading amount of the centrifuge solution passing through the macroporous adsorption resin 1 is 0.1 g to 0.5 g of crude drug per 1 mL column volume; The sample loading flow rate of the macroporous adsorption resin 1 is 1.0 to 4.0 times the column volume.

6. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step b, the weak base is any one of sodium carbonate, disodium hydrogen phosphate, ammonia water or sodium acetate.

7. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 6, characterized in that: The weak base solution is a disodium hydrogen phosphate solution, and the concentration of the disodium hydrogen phosphate solution is 2.0‰ to 4.0‰.

8. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step c, at least one of the following is met: Adjust the pH value of the alkaline eluent to 2-5; The model of the macroporous adsorption resin 2 is HPD-100, HPD-400, AB-8 or ADS-7; The loading amount of the centrifuge solution passing through the macroporous adsorption resin 2 is 0.1 g to 0.5 g of crude drug per 1 mL column volume; The loading flow rate of the macroporous adsorption resin 2 is 1.0 to 4.0 times the column volume; The volume concentration of the ethanol solution is 20% to 60%.

9. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step d, at least one of the following is satisfied: The alcohol eluate is concentrated to an extract concentration of 2.0 g crude drug / mL to 5.0 g crude drug / mL; The refrigeration time is 1 to 5 days; The drying temperature is 50-80°C.

10. The method for extracting and separating chlorogenic acid from Lonicera japonica according to claim 1, characterized in that: In step d, the solvent is any one of water, 30% volume concentration methanol solution, 50% volume concentration methanol solution, 70% volume concentration methanol solution or methanol.

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