Method for extracting crocin from gardenia processing residues

Abstract

The invention relates to a method for extracting crocin by using gardenia processing residues, which comprises the following steps of: firstly, drying, crushing and sieving residual gardenia meal after cold pressing gardenia fruits, and storing for later use; and mixing a deep eutectic solvent composed of an ammonium chloride hydrogen bond acceptor and a hydrogen bond donor according to a molar ratio, heating to be clear and transparent, cooling and standing to room temperature. And finally, weighing a certain amount of gardenia meal powder, putting the gardenia meal powder into a beaker, respectively mixing the gardenia meal powder with eight deep eutectic solvents according to a certain solid-liquid ratio, putting the mixture into a water bath, carrying out ultrasonic-assisted extraction, concentrating the obtained solution, and fixing the volume to obtain the crocin crude extract. Compared with a traditional method, the method has the advantages that the crocin extraction rate is high, the extraction solvent is green and environment-friendly, and the method is simple to operate, green, environment-friendly and efficient in extraction.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and separation of natural products, and particularly relates to a method for extracting and separating crocetin from gardenia meal by using a deep eutectic solvent. Background Art

[0002] Gardenia is a common shrub in the family Rubiaceae, mainly grown in tropical and subtropical regions such as China, Japan, Korea, India, and North America. Gardenia fruit is the dried ripe fruit of the gardenia plant, which is not only a raw material for natural dyes but also a traditional Chinese herbal medicine with anti-inflammatory, analgesic, antipyretic, anti-atherosclerotic and other effects. Gardenia is one of the first important plant resources for both medicine and food in China. During the processing of gardenia oil, a large amount of gardenia meal is produced. The produced gardenia meal can be used for the production of gardenia pigment. However, the traditional production of gardenia yellow pigment mainly relies on alcohol extraction. In order to reduce the environmental impact, enterprises need to consume a large amount of manpower and material resources in dealing with organic reagents, increasing the production cost. Therefore, choosing an efficient and green extraction method plays an important role in improving the quality and efficiency of the industrial utilization of gardenia fruit.

[0003] Crocin, also known as safranal glycoside, is one of the main bioactive components in gardenia, which has anti-cancer, anti-tumor and other effects. Crocin is the only water-soluble carotenoid existing in nature. Gardenia is a plant that contains a large amount of crocin besides saffron, and is an ideal source of crocin. Its chemical composition is a glycoside formed by crocetin and gentiobiose or glucose, and there are four structures in total, namely crocin-1, crocin-2, crocin-3 and crocin-4. Among them, the main component in gardenia fruit is crocin-1, accounting for more than 70% of the total crocin.

[0004] At present, the extraction method of crocin in gardenia mainly uses traditional organic solvents such as ethanol, methanol or acetone, which has an adverse impact on the environment. In recent years, natural deep eutectic solvents (NADES), as a green alternative to traditional solvents, have the advantages of good solubility, biocompatibility, low volatility, sustainability, low toxicity and easy production, and have been increasingly used to extract various bioactive compounds, including polyphenols, anthocyanins and carotenoids. NADES is synthesized by mixing primary metabolites or bio-renewable materials, and its melting point is lower than that of its components. One of the components serves as a hydrogen bond donor (HBD) (such as amino acids, carboxylic acids, sugars), and the other component serves as a hydrogen bond acceptor (HBA) (such as quaternary ammonium salts). Due to its composition, NADES usually has high viscosity, resulting in reduced mass transfer ability. However, by adding an appropriate amount of water to adjust the physicochemical properties of NADES and combining NADES with microwave-ultrasonic assisted extraction, this problem can be effectively avoided. Summary of the Invention

[0005] Technical problem to be solved: Aiming at the problems existing in the prior art, the present invention provides a method for extracting crocin from the residues of gardenia processing. The method provided by the present invention uses a deep eutectic solvent and a microwave-ultrasound combined assisted method to extract crocin from gardenia meal. This method is simple to operate, environmentally friendly, low-cost and highly efficient.

[0006] Technical solution: A method for extracting crocin from the residues of gardenia processing, the steps are as follows: (1) Preparation of gardenia meal powder: After cold pressing gardenia fruits, gardenia meal is obtained, dried and then pulverized and sieved for use; (2) Preparation of deep eutectic solvent: The deep eutectic solvent is composed of an ammonium chloride-based hydrogen bond acceptor HBA and a hydrogen bond donor HBD. The ammonium chloride-based hydrogen bond acceptor HBA is selected from at least one of choline chloride and betaine; The organic acid hydrogen bond donor HBD is selected from at least one of 1,2-propanediol, urea, malic acid, glucose, lactic acid and citric acid; Mix the ammonium chloride-based hydrogen bond acceptor HBA and the hydrogen bond donor HBD, heat to clear and transparent, and let it stand and cool to room temperature to obtain the deep eutectic solvent; (3) Extraction of crocin: Take the gardenia meal powder obtained in step (1) in a container, mix it with the deep eutectic solvent prepared in step (2), and carry out microwave-ultrasound combined assisted extraction to obtain crocin.

[0007] In step (2), the molar ratio of the ammonium chloride-based hydrogen bond acceptor HBA to the hydrogen bond donor HBD is 2:1 to 1:2.

[0008] In step (2), the mixing temperature of the ammonium chloride-based hydrogen bond acceptor HBA and the hydrogen bond donor HBD is 60-80 °C.

[0009] In step (3), the mass-volume ratio of gardenia meal powder to the deep eutectic solvent is 1:20 to 1:50 (g / mL).

[0010] In step (3), the microwave-assisted power is 40-80 Hz.

[0011] In step (3), the ultrasonic-assisted power is 100-200 W.

[0012] In step (3), the extraction time is 10-30 min.

[0013] It further includes step (4): After extraction, remove the solid residue by centrifugation or filtration to obtain a solution containing crocin.

[0014] It further includes step (5): Concentrate and / or purify the solution containing crocin obtained in step (4) to improve the purity and concentration of crocin.

[0015] The above purification step includes using one of chromatographic techniques, crystallization techniques or membrane filtration techniques to further improve the purity of crocin.

[0016] Beneficial effects: The raw materials used in the present invention are the by-products after cold pressing and extracting gardenia oil from gardenia fruits. After further extraction, they can "turn waste into treasure" and increase the added value of the by-products. The extraction technology used in the present invention is microwave-ultrasound combined assisted deep eutectic solvent extraction. Firstly, the combination of ultrasound and microwave can accelerate the destruction of plant cells, enable the extraction medium to directly enter the cells, strengthen the mass transfer rate between the plant matrix and the solvent, and thus improve the dissolution degree of active ingredients. Secondly, the extraction process is carried out at a lower temperature, avoiding the degradation of bioactive substances caused by high temperature. In the method of the present invention, the gardenia meal after cold pressing is dried and crushed and then added to the deep eutectic solvent for ultrasonic-assisted extraction of crocin, improving the utilization efficiency of processing residues. Description of the drawings

[0017] Figure 1 It is the HPLC chromatogram of the crocin control sample.

[0018] Figure 2 It is the HPLC chromatogram of the crude crocin extracts extracted by the deep eutectic solvent, pure water, ethanol and microwave-ultrasound combined assisted method in Examples 1-8 and Comparative Examples 1-3.

[0019] Figure 3 It is the content of crocin extracted by the deep eutectic solvent, pure water, ethanol and microwave-ultrasound combined assisted method in Examples 1-8 and Comparative Examples 1-3. Detailed implementation manners

[0020] To better understand the present invention, the following specific examples are used to further illustrate the present invention in detail. The specific examples described in the implementation manners of the invention are only illustrative descriptions of the specific implementation manners of the present invention, but not limitations on the present invention.

[0021] Example 1

[0022] The present invention provides a method for extracting crocin from gardenia processing residues, and the method comprises the following steps:

[0023] Step (1): Preparation of the sample: The gardenia meal remaining after cold pressing the gardenia fruits is dried in an oven at 60°C. The dried gardenia meal is crushed in a pulverizer, sieved, and stored for use.

[0024] Step (2): Preparation of the deep eutectic solvent: Choline chloride and 1,2-propanediol are mixed in a molar ratio of 1:2, heated and mixed at 80-90°C until clear and transparent, and cooled and allowed to stand at room temperature to obtain the deep eutectic solvent used for extraction.

[0025] Step (3): Extraction of crocin: Take gardenia meal powder in a beaker, mix it with a deep eutectic solvent according to a solid-liquid ratio of 1:20 - 1:50 (g / mL), perform microwave-assisted extraction at 40 - 80 Hz for 30 s, then perform ultrasonic-assisted extraction at 100 - 200 w for 10 - 30 min. Concentrate the obtained solution and make up the volume to 25 mL for later use.

[0026] Analysis conditions: Chromatographic column: C18 column (200×4.6 mm, 5 μm), column temperature: 30 °C; Mobile phase: acetonitrile (A) and 1% (v / v) formic acid aqueous solution (B), gradient elution: 0 - 10 min, 90 - 75% B; 10 - 15 min, 75 - 65% B; 15 - 25 min, 65 - 45% B; Flow rate: 1 mL / min; Detector: DAD, detection wavelength 440 nm.

[0027] Example 2

[0028] The present invention provides a method for extracting crocin from gardenia processing residues, and the method comprises the following steps:

[0029] Step (1): The same as above.

[0030] Step (2): Preparation of deep eutectic solvent: Mix choline chloride and urea according to a molar ratio of 1:2, heat and mix at 80 - 90 °C until clear and transparent, cool and let stand to room temperature to obtain the deep eutectic solvent for extraction.

[0031] Step (3): The same as above.

[0032] The HPLC conditions are the same as above.

[0033] Example 3

[0034] The present invention provides a method for extracting crocin from gardenia processing residues, and the method comprises the following steps:

[0035] Step (1): The same as above.

[0036] Step (2): Preparation of deep eutectic solvent: Mix choline chloride and malic acid according to a molar ratio of 1:1, heat and mix at 80 - 90 °C until clear and transparent, cool and let stand to room temperature to obtain the deep eutectic solvent for extraction.

[0037] Step (3): The same as above.

[0038] The HPLC conditions are the same as above.

[0039] Example 4

[0040] The present invention provides a method for extracting crocin from gardenia processing residues, and the method comprises the following steps:

[0041] Step (1): The same as above.

[0042] Step (2): Preparation of the deep eutectic solvent: Mix choline chloride and glucose in a molar ratio of 2:1, heat the mixture at 80 - 90 °C until it becomes clear and transparent, and then cool it to room temperature and let it stand still to obtain the deep eutectic solvent for extraction.

[0043] Step (3): The same as above.

[0044] The HPLC conditions are the same as above.

[0045] Example 5

[0046] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0047] Step (1): The same as above.

[0048] Step (2): Preparation of the deep eutectic solvent: Mix choline chloride and lactic acid in a molar ratio of 1:4, heat the mixture at 80 - 90 °C until it becomes clear and transparent, and then cool it to room temperature and let it stand still to obtain the deep eutectic solvent for extraction.

[0049] Step (3): The same as above.

[0050] The HPLC conditions are the same as above.

[0051] Example 6

[0052] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0053] Step (1): The same as above.

[0054] Step (2): Preparation of the deep eutectic solvent: Mix choline chloride and citric acid in a molar ratio of 1:1, heat the mixture at 80 - 90 °C until it becomes clear and transparent, and then cool it to room temperature and let it stand still to obtain the deep eutectic solvent for extraction.

[0055] Step (3): The same as above.

[0056] The HPLC conditions are the same as above.

[0057] Example 7

[0058] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0059] Step (1): The same as above.

[0060] Step (2): Preparation of the deep eutectic solvent: Mix betaine and 1,2 - propanediol in a molar ratio of 1:2, heat the mixture at 80 - 90 °C until it becomes clear and transparent, and then cool it to room temperature and let it stand still to obtain the deep eutectic solvent for extraction.

[0061] Step (3): The same as above.

[0062] The HPLC conditions are the same as above.

[0063] Example 8

[0064] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0065] Step (1): The same as above.

[0066] Step (2): Preparation of the deep eutectic solvent: Mix betaine and glucose in a molar ratio of 2:1, heat the mixture at 80 - 90 °C until it becomes clear and transparent, and cool and stand it to room temperature to obtain the deep eutectic solvent for extraction.

[0067] Step (3): The same as above.

[0068] The HPLC conditions are the same as above.

[0069] Comparative Example 1

[0070] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0071] Step (1): The same as above.

[0072] Step (2): Preparation of the extraction solvent: The extraction solvent is pure water.

[0073] Step (3): Extraction of crocin: Take gardenia meal powder in a beaker, mix it with water at a solid-liquid ratio of 1:20 - 1:50 (g / mL), place it in a water bath at 40 - 60 °C, and extract for 2 h. The obtained solution is concentrated and then fixed in volume to 25 mL for use.

[0074] The HPLC conditions are the same as above.

[0075] Comparative Example 2

[0076] The present invention provides a method for extracting crocin from the residues of gardenia processing, and the method comprises the following steps:

[0077] Step (1): The same as above.

[0078] Step (2): Preparation of the extraction solvent: The extraction solvent is 60% ethanol. Take 60 mL of absolute ethanol in a 100 mL volumetric flask, and add ultrapure water to make up the volume to 100 mL.

[0079] Step (3): Extraction of crocin: Take gardenia meal powder in a beaker, mix it with water at a solid-liquid ratio of 1:20 - 1:50 (g / mL), place it in a water bath at 40 - 60 °C, and extract for 2 h. The obtained solution is concentrated and then fixed in volume to 25 mL for use.

[0080] HPLC conditions were the same as above.

[0081] Comparative Example 3

[0082] Step (1): Same as above.

[0083] Step (2): Preparation of extraction solvent: The extraction solvent is pure water.

[0084] Step (3): Extraction of crocin: Take the gardenia meal powder in a beaker, mix it with pure water at a solid-liquid ratio of 1:20 to 1:50 (g / mL), perform 40-80 Hz microwave-assisted extraction for 30 seconds, and then perform 100-200 w ultrasonic-assisted extraction for 10-30 minutes. The resulting solution is concentrated and fixed to 25 mL for standby use.

[0085] HPLC conditions were the same as above.

[0086] Figure 1 This is the HPLC chromatogram of crocin standard, with a retention time of 3.028 min. Figure 2 The analysis showed that all extraction methods were able to extract crocin, among which the peak areas of extracts I, J and K were relatively large. Figure 3 It can be seen that compared with the traditional method (extract A, extract B and extract C) in the comparative example, the extract J, extract I and extract K extracted by deep eutectic solvent have higher crocin content, followed by choline chloride-1,2-propylene glycol, choline chloride-urea and choline chloride-lactic acid. Among them, the crocin content extracted by the combination of choline chloride-1,2-propylene glycol is the highest. Therefore, microwave-ultrasound combined assisted deep eutectic solvent can be used to extract crocin from gardenia meal, realize green extraction, reduce environmental pollution risks, and at the same time, improve crocin extraction efficiency, which is an efficient and environmentally friendly extraction method.

Claims

1. A method for extracting crocin from the residues of gardenia processing, characterized in that, The steps are as follows: (1) Preparation of gardenia meal powder: After cold pressing gardenia fruits, gardenia meal is obtained, dried, pulverized, and sieved for later use; (2) Preparation of deep eutectic solvent: The deep eutectic solvent is composed of an ammonium chloride-based hydrogen bond acceptor HBA and a hydrogen bond donor HBD. The ammonium chloride-based hydrogen bond acceptor HBA is selected from at least one of choline chloride and betaine; the organic acid hydrogen bond donor HBD is selected from at least one of 1,2-propanediol, urea, malic acid, glucose, lactic acid, and citric acid; The ammonium chloride-based hydrogen bond acceptor HBA and the hydrogen bond donor HBD are mixed, heated to clear and transparent, and allowed to stand and cool to room temperature to obtain a deep eutectic solvent; (3) Extraction of crocin: Take the gardenia meal powder obtained in step (1) in a container, mix it with the deep eutectic solvent prepared in step (2), and perform microwave-ultrasound combined assisted extraction to obtain crocin.

2. The method for extracting crocin from the processing residues of gardenia according to claim 1, characterized in that, In step (2), the molar ratio of the ammonium chloride-based hydrogen bond acceptor HBA to the hydrogen bond donor HBD is 2:1 to 1:

2.

3. The method for extracting crocin from gardenia processing residues according to claim 1, characterized in that, In step (2), the mixing temperature of the ammonium chloride-based hydrogen bond acceptor HBA and the hydrogen bond donor HBD is 60 - 80 °C.

4. The method for extracting crocin from gardenia processing residues according to claim 1, characterized in that, In step (3), the mass-volume ratio of the gardenia meal powder to the deep eutectic solvent is 1:20 - 1:50 (g / mL).

5. The method for extracting crocin from gardenia processing residues according to claim 1, wherein In step (3), the microwave-assisted power is: 40 - 80 Hz.

6. The method for extracting crocin from the processed residues of gardenia according to claim 1, characterized in that, In step (3), the ultrasound-assisted power is: 100 - 200 W.

7. The method for extracting crocin from the processing residues of gardenia according to claim 1, characterized in that, In step (3), the extraction time is 10 - 30 min.

8. The method for extracting crocin from gardenia processing residues according to claim 1, characterized in that, It further includes step (4): After extraction, solid residues are removed by centrifugation or filtration to obtain a solution containing crocin.

9. The method for extracting crocin from the processing residues of gardenia according to claim 8, characterized in that, It further includes step (5): The solution containing crocin obtained in step (4) is concentrated and / or purified to improve the purity and concentration of crocin.

10. The method for extracting crocin from gardenia processing residues according to claim 9, characterized in that, The purification step includes using one of chromatographic techniques, crystallization techniques, or membrane filtration techniques to further improve the purity of crocin.

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