Method for extracting honeysuckle chlorogenic acid by using disubstituted chitosan oligosaccharide in cooperation with deep-eutectic solvent

Abstract

The invention relates to a method for extracting chlorogenic acid from honeysuckle by using disubstituted chitosan oligosaccharide and a deep-eutectic solvent, which comprises the following steps: extracting honeysuckle by using a deep-eutectic mixed system by adopting a two-stage extraction process, then continuously extracting by using an ethanol solution, and carrying out resin separation and elution after extraction to obtain chlorogenic acid, wherein the eutectic mixing system comprises disubstituted chitosan oligosaccharide, water and acetic acid; the structural formula of the disubstituted chitosan oligosaccharide is shown in the specification. During extraction, a two-stage extraction process is adopted, firstly, a water-acetic acid system containing a small amount of TACO is used for treatment, and due to the fact that acetic acid is high in acidity and large in amount, ionization of carboxyl of carboxypropionyl is avoided, hydrogen on the carboxyl can better play a role of a hydrogen bond donor, and meanwhile cross-linked precipitation caused by positive and negative charge attraction between TACO is inhibited; the permeability of the cell walls of the honeysuckle is improved, the problem of insufficient permeability of the cell walls in the traditional process is solved, and then the ethanol solution is used for extraction, so that the effect of extraction at low temperature is realized, and the difficulty of post-treatment is also reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extraction methods of honeysuckle, and in particular to a method for extracting chlorogenic acid from honeysuckle by using double-substituted chitooligosaccharides in cooperation with a deep eutectic solvent. BACKGROUND

[0002] Honeysuckle (Lonicera japonica Thunb.) is an important traditional Chinese medicinal material with multiple pharmacological activities such as clearing heat and detoxifying, antibacterial and anti-inflammatory. One of its main active components is chlorogenic acid, which is a phenolic acid compound with significant antioxidant, anti-inflammatory, antibacterial, antiviral, hypoglycemic and potential antitumor activities. The content of chlorogenic acid is one of the key indicators for evaluating the quality of honeysuckle and its products.

[0003] At present, the commonly used methods for extracting chlorogenic acid from plant materials such as honeysuckle mainly include organic solvent extraction, water extraction, supercritical fluid extraction, etc. Among them, the organic solvent extraction method belongs to the traditional extraction method, which mainly uses organic solvents such as methanol, ethanol, acetone as extractant for extraction. This method has relatively high extraction rate, but has the following significant shortcomings: (1) a large amount of flammable, volatile and toxic organic solvents are used, which has safety hazards and environmental pollution problems; (2) solvent residues may affect product safety, especially for food and drug applications; (3) some organic solvents (such as methanol) have high toxicity, which need to be strictly controlled; (4) high-temperature extraction may cause partial degradation of heat-sensitive chlorogenic acid; (5) in order to avoid decomposition at high temperature, the traditional ethanol extraction method usually needs to add additional protective agents (such as vitamin C). Water extraction uses water as the solvent for extraction, which is safe and low in cost. However, its main shortcomings are: (1) the solubility of chlorogenic acid in water is limited, which leads to the extraction efficiency usually lower than that of organic solvent method; (2) the extraction selectivity is poor, which will simultaneously dissolve a large amount of impurities such as polysaccharides, proteins and pigments, making it difficult for subsequent separation and purification, increasing cost and complexity; (3) the extraction solution is prone to spoilage and deterioration. Supercritical fluid extraction uses non-polar supercritical CO2 for extraction, which is green, solvent-free and low in operating temperature. However, chlorogenic acid is a compound with strong polarity, and its solubility in liquid CO2 is very low, usually requiring the addition of a polar entrainer (such as ethanol, methanol), which to some extent weakens its "green" advantage, and the high investment and operating cost of the equipment limits its large-scale application.

[0004] In recent years, deep eutectic solvent (DES) as a new type of green solvent has been widely concerned. DES refers to a stable solvent formed by the intermolecular hydrogen bond association of two or more substances, which is generally liquid at room temperature. It is usually formed by hydrogen bond donor (HBD, such as organic acid, polyol, amide, etc.) and hydrogen bond acceptor (HBA, such as choline chloride) through hydrogen bond interaction. DES has many advantages such as simple preparation, cheap and easily available raw materials, low or no toxicity, biodegradability, low vapor pressure, non-volatility, strong structure designability and so on. However, there are some key difficulties in the practical extraction application of DES: (1) The viscosity of many efficient DES is much higher than that of traditional organic solvents and water, which leads to large mass transfer resistance, poor permeability, limits the diffusion of DES into plant tissue and the dissolution efficiency of target components, and affects the extraction speed and yield; (2) The hydrophilic DES is easy to dissociate in water, which limits the possibility of reducing the viscosity of the system by dilution with water; (3) The accumulation of impurities leads to the decrease of the efficiency after circulation, and there is a lack of industrial regeneration process. Therefore, the low eutectic solvent extraction technology has not yet formed industrial application. In theory, as long as the conditions for forming a certain hydrogen bond system are met, it is possible to realize the high extraction efficiency of the deep eutectic system. Therefore, it is of great significance to develop efficient, environmentally friendly and highly selective chlorogenic acid extraction technology based on DES for improving the utilization value of honeysuckle resources and ensuring the quality of medicines / health products. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application uses chitooligosaccharide as the basic unit and cooperates with the low eutectic solvent system to realize the efficient extraction of chlorogenic acid in honeysuckle.

[0006] To achieve the above object, the present application provides the following technical scheme: A method for extracting chlorogenic acid in honeysuckle by using double-substituted chitooligosaccharide in cooperation with low eutectic solvent, which is a two-stage extraction process. The honeysuckle is first extracted with a low eutectic mixed system, and then extracted with an ethanol solution. After extraction, the resin is separated and eluted to obtain chlorogenic acid. The low eutectic mixed system comprises double-substituted chitooligosaccharide, water and acetic acid. The structural formula of the double-substituted chitooligosaccharide (TACO) is , wherein n = 8-14, and n + m ≤ 20.

[0007] Further, the mass-volume ratio of the double-substituted chitooligosaccharide, water and acetic acid in the low eutectic mixed system is 1g:1-2ml:2-4ml.

[0008] Further, the solid-liquid ratio of the honeysuckle to the low eutectic mixed system is 1g:2-4ml.

[0009] Further, the solid-liquid ratio of the honeysuckle to the ethanol solution is 1g:10-20ml, and the concentration of the ethanol solution is 50-70%.

[0010] Further, the method comprises the following steps: 1) Mix the measured double-substituted chitooligosaccharide, water and acetic acid, heat to 60-90℃, and cool to obtain a eutectic mixture; 2) Add the measured honeysuckle powder to the eutectic mixture, and ultrasonically extract at a certain temperature for 20-60 min; 3) Add a certain amount of ethanol solution to step 2), and continue to extract for 20-60 min; 4) Centrifuge the mixture of step 3), separate the supernatant, and remove the lower solid; 5) Concentrate the supernatant under reduced pressure, pass through a macroporous resin column, elute with water and 30% ethanol gradient, collect the eluate, concentrate under reduced pressure, extract with ethyl acetate, and finally concentrate and crystallize to obtain the chlorogenic acid product.

[0011] Further, the extraction temperature of step 2) is 30-50℃.

[0012] The beneficial effects of the present application are: two-stage extraction process is adopted during extraction, first, the water-acetic acid system containing a small amount of TACO is used for treatment, because the acetic acid is strongly acidic and in large amount, the carboxyl group ionization of carboxypropionyl is avoided, so that the hydrogen on the carboxyl group can better play the role of hydrogen bond donor, and the crosslinking and precipitation of TACO due to the attraction of positive and negative charges is inhibited; the permeability of honeysuckle cell wall is improved, solving the problem of insufficient cell wall permeability in traditional process, and then the ethanol solution is used for extraction, not only realizing the effect of low-temperature extraction, but also reducing the difficulty of post-treatment. Because the water-acetic acid system is a low-boiling-point liquid, it is easy to recover and separate during post-treatment; and TACO itself is an oligosaccharide with large molecular weight, which is very easy to separate from small molecular extracts (chlorogenic acid) during post-treatment separation (such as macroporous resin treatment). BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0014] Figure 1 It is an infrared spectrum of double-substituted chitooligosaccharide (TACO) (50%).

[0015] Figure 2 It is an ultraviolet scanning spectrum of chlorogenic acid standard.

[0016] Figure 3 It is an ultraviolet scanning spectrum of the extraction centrifugal liquid of examples 1-4.

[0017] Figure 4 This is a standard curve for the determination of chlorogenic acid by ultraviolet spectrophotometry in an aqueous system. Detailed Implementation

[0018] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] Example: A method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent. A two-stage extraction process was used to extract honeysuckle first using a eutectic mixture system, followed by extraction with an ethanol solution. After extraction, chlorogenic acid was obtained by resin separation and elution. The eutectic mixture system comprised a disubstituted chitosan oligosaccharide, water, and acetic acid. The disubstituted chitosan oligosaccharide (TACO) has the following structural formula: , where n = 8~14, n + m ≤20.

[0021] The molecular reaction formula for synthesizing chitosan oligosaccharide derivatives is as follows: Step 1) Dissolve 1610 mg of chitosan oligosaccharide in 40 ml of water, add 500 mg of succinic anhydride, and react at room temperature for 3-8 h. Then add the filtrate from Step 1) and continue the reaction at room temperature for 4-8 h.

[0022] Step 2) Dissolve 472.5 mg chloroacetic acid and 700 μl triethylamine in DMF, then add 615 μl of trimethylacetyl chloride in DMF dropwise. Stir for 2–8 h until the reaction is complete. Filter to remove the precipitate and keep the filtrate for later use.

[0023] Step 3) Add 860 μl of trimethylamine solution to the reaction solution in Step 2), heat to 50~80℃ and react for 3~8h, cool and adjust pH to 4~6.

[0024] Step 4) Add ethanol to the reaction solution in Step 3), allow it to precipitate completely, and then centrifuge to obtain the product TACO.

[0025] The crude chitosan oligosaccharide derivative was dissolved in an appropriate amount of water, and n-propanol was added for secondary precipitation. After centrifugation and vacuum drying, a product with high purity was obtained.

[0026] The infrared spectrum of the BACO (50%) prepared in this embodiment is attached. Figure 1 As shown.

[0027] In the appendix Figure 1 Medium at 3294 cm -1 The broad peaks nearby are mainly due to the stretching vibrations of the OH group on the hydroxyl group and the NH group on the amide group of glucosamine, at 2930 cm⁻¹. -1 ~2810 cm -1 The peak value of the stretching vibration of the CH bond on the carbon-carbon double bond is located nearby; 1632 cm⁻¹ -1 ~1597 cm -1 The absorption at this point is mainly formed by the conjugation of the stretching vibration peak of the amide bond and the characteristic absorption peak of the benzene ring skeleton vibration.

[0028] Elemental analysis revealed that the content of each element in TACO was: C 49.0%, N 8.57%, and O 42.4%, which is basically consistent with the theoretical results (C 50.1%, N 8.1%, and O 41.8%).

[0029] In this invention, a betaine acyl group is introduced onto the chitosan oligosaccharide, which can act as a highly efficient hydrogen bond acceptor; simultaneously, the carboxyl group (-COOH) of the introduced carboxypropyl group mainly serves as a hydrogen bond donor. In eutectic systems (such as water-acetic acid), when the proportion of acetic acid is much greater than that of water, the hydrogen ionization of acetic acid and the carboxyl group of the carboxypropyl group is very low, and they exist almost in molecular form. Although hydrogen bonds exist between acetic acid and water (forming dimers and chain structures), water and acetic acid molecules are small and easily flowable, making it impossible to form a strong hydrogen bond system. When TACO is added, it is generally believed that the chloride ions of the betaine acyl group can form a strong hydrogen bond network with multiple hydrogen bond donors (such as -COOH), thereby driving the formation of a eutectic mixture. Since the positions of the betaine acyl group and carboxypropyl group on the chitosan oligosaccharide backbone are relatively fixed, within a certain concentration range, a strong hydrogen bond system similar to a deep eutectic system may be formed. This system can disrupt the inter-chain network of cellulose through competitive hydrogen bonding. At the same time, its own groups (carbonyl, amino, hydroxyl, etc.) can form a stable "solventization" layer on the cellulose surface, thereby destroying the cellulose structure (dissolving hemicellulose), increasing cell wall permeability, and promoting the dissolution of small molecule extracts.

[0030] A two-stage extraction process is used for honeysuckle. First, extraction is performed using a eutectic mixture system. Because the viscosity of the eutectic mixture system is very low, it helps the strong hydrogen bond system formed by TACO to act on the cell wall of honeysuckle. After destroying the cellulose structure, the cell permeability increases. Then, extraction is performed with an ethanol solution, supplemented by an ultrasonic system, which can achieve efficient extraction at low temperature. By repeating the ethanol solution extraction multiple times, the chlorogenic acid contained in honeysuckle can be completely extracted.

[0031] The following specific examples evaluate the performance of the disubstituted chitosan oligosaccharide synergistic eutectic solvent in the extraction of chlorogenic acid from honeysuckle.

[0032] Example 1: Step 1) Weigh 0.15 g TACO, add 1 mL water and 3 mL acetic acid, heat to 80℃, keep warm for 1 h, and cool to obtain the eutectic flux.

[0033] Step 2) Weigh 1 g of honeysuckle powder, add a eutectic agent, and ultrasonically extract at 45℃ for 30 min.

[0034] Step 3) Add 10 mL of 60% ethanol solution to Step 2) and continue extraction for 60 min.

[0035] Step 4) Centrifuge the mixture from Step 3) and separate the supernatant (or add a certain amount of ethanol solution in small amounts multiple times for extraction, and finally combine the centrifuged liquids to obtain the final extract). Observe the absorption of the ultraviolet spectrum.

[0036] Example 2: Step 1) Weigh 1 g of honeysuckle powder, add 1 mL of water and 3 mL of acetic acid solution, and extract by ultrasonication at 45℃ for 30 min.

[0037] Step 2) Add 10 mL of 60% ethanol solution to Step 1) and continue extraction for 60 min.

[0038] Step 3) Centrifuge the mixture from Step 2) to separate the supernatant and scan the UV absorption spectrum.

[0039] Example 3: Step 1) Weigh 2 g of choline chloride, add 4 mL of glycerol, heat to 80℃ and keep warm for 1 hour. After cooling, a clear and transparent solution is obtained.

[0040] Step 2) Weigh 1 g of honeysuckle powder, add it to the solution obtained in Step 1), and extract it by ultrasonication at 45℃ for 30 min.

[0041] Step 3) Add 10 mL of 60% ethanol solution to Step 2) and continue extraction for 60 min.

[0042] Step 4) Centrifuge the mixture from Step 3) to separate the supernatant and scan the absorption spectrum using ultraviolet light.

[0043] Example 4: Step 1) Weigh 1 g of honeysuckle powder and 10 mL of 60% ethanol solution, and extract by ultrasonication at 45℃ for 60 min.

[0044] Step 2) Centrifuge the mixture from Step 1) to separate the supernatant and scan the absorption spectrum using ultraviolet light.

[0045] Appendix Figure 2 This is the UV scan of chlorogenic acid standard in aqueous solution, with λ=324 nm as the characteristic absorption peak.

[0046] Appendix Figure 3 The images show the UV-Vis absorption spectra of the extraction centrifuges from Examples 1-4. The absorbance at λ=324 nm is as follows: a(TACO + acetic acid + water): 1.029; b(choline chloride + glycerol): 0.975; c(acetic acid + water): 0.925; d(ethanol): 0.629. This indicates that adding a small amount of TACO is sufficient to effectively extract chlorogenic acid under acetic acid conditions. Under similar conditions, the extraction efficiency of the acetic acid-TACO-water system is approximately 5.5% higher than the traditional choline chloride-glycerol system, approximately 10% higher than the acetic acid + water system, and approximately 63.6% higher than the traditional ethanol system. These data indicate that TACO significantly improves extraction efficiency in an acetic acid-water medium. This may be related to the formation of strong hydrogen bonds in the system. Because the molecular structure of TACO is very similar to that of cellulose, it can form hydrogen bonds with groups on the surface chains of cellulose through multi-site polar groups, thus adsorbing onto the surface and forming a certain degree of "solventization." Meanwhile, the chloride ions of the betaine chloride structure are in a free state in the medium and can form strong hydrogen bonds with the carboxyl groups of acetic acid or nearby chitosan oligosaccharides. When these strong hydrogen bonds encounter the hydrogen bonds between cellulose molecular chains... Under specific structural guidance, the hydrogen bonds can be dissociated, resulting in a loose cellulose molecular chain and increased cell wall permeability. Therefore, its extraction efficiency is higher than that of the acetic acid-water system. Compared with the traditional DES system and choline chloride-glycerol system, the TACO system has a much lower viscosity and acts on cell wall cellulose much more efficiently, thus also having a higher extraction efficiency. Compared with the traditional ethanol system, ethanol itself cannot destroy cell wall cellulose and can only pass through the cell wall. Chlorogenic acid encounters greater resistance during the process of passing through the cell wall, thus resulting in a lower extraction efficiency.

[0047] The above-mentioned centrifuged liquid was post-treated to obtain high-quality chlorogenic acid, and the steps are as follows: 1) After extracting the centrifuged sediment twice with 60% ethanol solution, combine the centrifuged liquids and concentrate them under reduced pressure at 45°C in a rotary evaporator until there is no alcohol odor. Determine the chlorogenic acid content using ultraviolet spectrophotometry, control the chlorogenic acid content to be between 4 and 6 mg / mL, and calculate the total amount of chlorogenic acid in the crude product.

[0048] 2) Take macroporous resin (NKA-9) at a ratio of 40 mg chlorogenic acid / g resin, pack the chromatography column using the wet method, soak the resin in 95% ethanol for 24 h, pass the column through deionized water until the effluent has no alcohol odor, load the crude solution from step 1 onto the column, control the column outlet flow rate at 1~2 BV / h (BV: the volume of resin packed in the chromatography column), elute with water and 30% ethanol solution sequentially, collect the effluent in separate tubes, and stop when the concentration of chlorogenic acid in the column outlet effluent is close to 0.

[0049] 3) Combine the collection tubes containing chlorogenic acid in the effluent, concentrate until there is no alcohol odor, combine the concentrates, add ethyl acetate at a ratio of 1:0.4 for extraction, repeat three times, combine the ethyl acetate solutions, concentrate and crystallize to obtain the chlorogenic acid product.

[0050] Appendix Figure 4 The standard curve for determining chlorogenic acid by ultraviolet spectrophotometry in an aqueous system is shown in Table 1. Based on this, the purity of the chlorogenic acid product is determined and recorded.

[0051] Table 1 As shown in Table 1, the purity differences among the various examples are relatively small, with Example 4 exhibiting the highest purity. This is related to cell permeability; when cell permeability is high, more substances permeate through the cell wall, thus decreasing the purity of chlorogenic acid in the extract. The ethanol system has a relatively small effect on the cell wall, resulting in less permeation and consequently, a relatively high purity after extraction. This indicates that the extraction method presented in this study not only achieves low-temperature extraction of chlorogenic acid but also further improves extraction efficiency while preserving the original purity of the extracted chlorogenic acid.

[0052] Five batches of honeysuckle powder were weighed repeatedly and extracted according to the steps in Example 2. The absorbance of the extract was measured and the results are recorded in Table 2.

[0053] Table 2 As shown in Table 2, the data of the five samples are basically consistent, with an average of 92.68%, s=0.463%, and CV≈0.5%<1%, indicating that the two-stage extraction process of the present invention has good repeatability and good process stability.

[0054] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent, characterized in that, The extraction process employs a two-stage extraction method. First, honeysuckle is extracted using a eutectic mixture system, followed by further extraction with an ethanol solution. After extraction, resin separation and elution yield chlorogenic acid. The eutectic mixture system comprises disubstituted chitosan oligosaccharides, water, and acetic acid. The structural formula of the disubstituted chitosan oligosaccharides is as follows: , where n = 8~14, n + m≤20.

2. The method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent according to claim 1, characterized in that, The mass-to-volume ratio of the disubstituted chitosan oligosaccharide, water, and acetic acid in the eutectic mixture is 1g:1~2ml:2~4ml.

3. The method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent according to claim 1, characterized in that, The ratio of honeysuckle to eutectic mixture is 1g:2~4ml.

4. The method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent according to claim 1, characterized in that, The ratio of honeysuckle to ethanol solution in the ethanol solution is 1g:10~20ml; the concentration of the ethanol solution is 50~70%.

5. The method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent according to claim 1, characterized in that, The method includes the following steps: 1) Mix the measured amount of disubstituted chitosan oligosaccharide, water, and acetic acid, heat to 60~90℃, and cool to obtain a eutectic mixture; 2) Add the measured amount of honeysuckle powder to the eutectic mixture and extract it by ultrasonic extraction at a certain temperature for 20-60 minutes; 3) Add a certain amount of ethanol solution to step 2) and continue extraction for 20-60 minutes; 4) Centrifuge the mixture from step 3) to separate the supernatant and the lower solids. 5) The supernatant was concentrated under reduced pressure, passed through a macroporous resin column, eluted with a gradient of water and 30% ethanol, the eluent was collected, concentrated under reduced pressure, extracted with ethyl acetate, and finally concentrated and crystallized to obtain chlorogenic acid product.

6. The method for extracting chlorogenic acid from honeysuckle using a disubstituted chitosan oligosaccharide and a eutectic solvent according to claim 5, characterized in that, The extraction temperature in step 2) is 30~50℃.

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