A method for preparing kynurenic acid from potato peel

Abstract

The application discloses a method for extracting kynurenic acid from potato peel, which comprises the following steps: freeze-drying and crushing potato peel to obtain potato peel powder; extracting the potato peel powder with water and ultrasonic, resolubilizing the crude extract SPUD with water, adding ethanol, storing at 4 DEG C for 12 hours, and removing the precipitate by centrifugation; separating and purifying the supernatant by Sephadex G-25, collecting 106 tubes of fractions, combining 9 components by TLC analysis, and obtaining component SPUD-5; adding acetic acid to the component SPUD-5, adding cation exchange resin for adsorption, eluting the adsorbed cation exchange resin with water, and obtaining component SPUD-5-6; taking the component SPUD-5-6, adsorbing with a macroporous resin, eluting the adsorbed macroporous resin with water, and obtaining kynurenic acid. The method separates kynurenic acid from potato peel for the first time, extracts kynurenic acid from potato waste, and can produce kynurenic acid and reduce environmental pollution.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and specifically to a method for preparing kynurenic acid from potato peel. Background Technology

[0002] Kynuric acid (KYNA) is the active metabolite of tryptophan. It was first demonstrated in urine by Liebig in 1853. KYNA is an endogenous antagonist of ionotropic glutamate receptors and α-7 nicotinic acetylcholine receptors, possessing anticonvulsant and neuroprotective activities, and it also regulates neurotransmission in the brain. Furthermore, according to literature, KYNA exhibits anti-ulcer and antioxidant properties, demonstrates anti-inflammatory properties in experimentally induced colonic obstruction and antagonizes intestinal hypermotility, reducing motility and inflammatory activation in the early stages of acute experimental colitis in rats.

[0003] Related technologies, such as Chinese invention patent CN 105801480 B, provide a method for preparing kynurenic acid from horseshoe crab tail, which is the first time that kynurenic acid has been isolated from horseshoe crab tail. Currently, there are no reports on the extraction of kynurenic acid from plants. Summary of the Invention

[0004] To address the above problems, embodiments of the present invention provide a method for extracting kynurenic acid from potato peels in a first aspect, comprising the following steps:

[0005] Step 1: Freeze-dry and pulverize the potato peels to obtain potato peel powder;

[0006] Step 2: Soak potato peel powder in water and extract with ultrasonic assistance. Repeat the extraction 3 times, combine the extracts, concentrate under reduced pressure to obtain crude extract SPUD.

[0007] Step 3: Redissolve the crude extract SPUD in water, add ethanol, store at 4°C for 12 hours, and centrifuge to remove the precipitate;

[0008] Step 4: Concentrate the supernatant to remove ethanol, redissolve in water, and separate and purify using Sephadex G-25 at a flow rate of 0.25 mL / min with water as the eluent. Collect each fraction using an automatic collector, collecting one tube every 30 min, for a total of 106 tubes. After TLC analysis, 9 components were obtained. The fractions from tubes 46-63 were combined and then concentrated under reduced pressure to obtain component SPUD-5.

[0009] Step 5: Take component SPUD-5, redissolve it in water, acidify it with acetic acid, add it to a cation exchange resin for adsorption, wash the adsorbed cation exchange resin with water, collect the aqueous solutions, combine them, evaporate the solvent to dryness, and obtain component SPUD-5-6.

[0010] Step 6: Take component SPUD-5-6, redissolve it in water, add it to macroporous resin for adsorption, and elute the adsorbed macroporous resin with water at a flow rate of 0.5-1 mL / min. Collect the components from 106 to 138 min to obtain kynurenic acid.

[0011] According to an embodiment of the present invention, a method for preparing kynurenic acid from dried tangerine peel is disclosed. This method extracts kynurenic acid from potato peel powder via water extraction and then purifies it using Sephadex G-25, ion exchange resin, and macroporous resin. This method is the first to isolate kynurenic acid from potato peel, utilizing potato waste (potato peel) for extraction. It can both produce kynurenic acid and reduce environmental pollution. Kynurenic acid is closely related to various neuropsychiatric disorders, renal failure, and other chronic malignant diseases, and therefore has high utilization value.

[0012] Optionally, in step 2, potato peel powder and water are added at a material-to-liquid ratio of 1:10, and ultrasonic extraction is performed for 1 hour.

[0013] Optionally, in step 3, add twice the volume of the concentrate containing 80% ethanol.

[0014] Optionally, in step 5, the cation exchange resin is a Dowex 50 ion exchange resin packed column.

[0015] In a second aspect, embodiments of the present invention provide the method described above for preparing kynurenic acid from potato peel to obtain kynurenic acid.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] Figure 1 The HPLC chromatogram of compound SPUD-5-6-6 according to an embodiment of the present invention is shown.

[0018] Figure 2 For the compound SPUD-5-6-6 according to embodiments of the present invention 1 H-NMR spectrum;

[0019] Figure 3 For the compound SPUD-5-6-6 according to embodiments of the present invention 13 C-NMR spectrum;

[0020] Figure 4 The results of thin-layer chromatography according to the present invention are shown; wherein, the left (developing solvent chloroform: methanol = 3:1) and the right (developing solvent chloroform: methanol = 5:1) are developed by ultraviolet analysis. Detailed Implementation

[0021] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0022] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0023] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0024] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0025] Example 1

[0026] A method for extracting kynurenic acid from potato peel includes the following steps:

[0027] Step 1: Freeze-dry the potato peels in a freeze dryer, then pulverize them at room temperature using a high-speed grinder to obtain potato peel powder. Store the powder in a sealed bag in a desiccator.

[0028] Step 2: Weigh 100g of potato peel powder and place it in a 2L conical flask. Add 1000mL of distilled water at a material-to-liquid ratio of 1:10. Extract with ultrasonic assistance for 1 hour. Repeat the extraction three times. Combine the solutions and evaporate the solvent using a rotary evaporator to obtain the crude extract SPUD. Store at 4℃ for later use.

[0029] Step 3: Redissolve the crude potato peel extract SPUD in water, add an appropriate amount of 80% ethanol, store at 4°C for 12 hours, and centrifuge to remove the precipitate.

[0030] Step 4: Concentrate the supernatant under reduced pressure to remove ethanol, redissolve in water, and purify using Sephadex G-25 at a flow rate of 0.25 mL / min with water as the eluent. Collect each fraction using an automatic collector, collecting one tube every 30 min, for a total of 106 tubes. TLC analysis yielded 9 components. The fractions from tubes 46-63 were combined and then concentrated under reduced pressure to obtain component SPUD-5.

[0031] Specifically, the thin-layer chromatography (TLC) method used is as follows: 1. Activate the GF254 silica gel plate in a 110℃ oven for 2-3 hours, and after it cools naturally to room temperature, remove it and place it in a desiccator for later use; 2. Use a glass capillary tube to draw an appropriate amount of sample and spot it on the baseline of the silica gel plate, marking the lower end of the sample spot with a pencil (the distance between the baseline of the silica gel plate and the bottom of the plate should be 0.8-1 cm, and the distance between two sample spots should not be less than 0.8 cm), and dry the solvent with a blower; 3. Place the chromatography plate in a chromatography tank containing chromatography solvent and develop it. When the solvent front reaches about 1 cm from the top of the thin-layer plate, remove it with tweezers and dry the solvent; 4. Develop the color using a UV analyzer and other colorimetric reagents (iodine colorimetry, sulfuric acid plate baking method, etc.). Figure 4 As shown.

[0032] The steps for separation and purification using Sephadex G-25 are as follows: 1. When loading the sample, adjust the tightness of the stopcock at the bottom of the column to control the eluent flow rate; 2. When the eluent level in the column is equal to the gel layer, use a dropper to add the sample along the column wall. Once the sample layer has descended to the level of the gel layer, wash the sample vial with a small amount of eluent and add the washing solution along the column wall into the gel column. Repeat the washing 2-3 times, then add a reservoir bulb; 3. Use water as the eluent, control the elution rate, collect the eluent fractions using an automatic collector, and combine the fractions according to thin-layer chromatography analysis.

[0033] Step 5: Take component SPUD-5, add an appropriate amount of water to redissolve it, add an appropriate amount of acetic acid to acidify it, add it to the cation exchange resin for adsorption, wash the adsorbed cation exchange resin with water, collect the aqueous solutions, combine them, evaporate the solvent to dryness, and obtain component SPUD-5-6.

[0034] Step 6: Take component SPUD-5-6, add an appropriate amount of water to redissolve it, add macroporous resin for adsorption, and wash the adsorbed macroporous resin with water at a flow rate of 0.5-1 mL / min. Collect the components from 106 to 138 min to obtain compound SPUD-5-6-6. The compound weighs 9.8 mg and has a purity of 93%.

[0035] Compound SPUD-5-6-6 is a white powder. HPLC analysis was performed using the following parameters: HPLC model: Agilent 1260 Infinity II; detector: 1260 DADWR; column: H&E-SPODS-TA column, 4.6 × 250 mm, s-5 μm; liquid phase conditions: mobile phase: chromatographic grade methanol and aqueous phase with 0.1% trifluoroacetic acid added; detection time: 30 min; flow rate: 1 mL / min; column temperature: 35 ℃; injection volume: 20.0 μL; detection wavelength: 238 nm.

[0036] The results are as follows Figure 1 As shown, Figure 1 The high-performance liquid chromatogram of compound SPUD-5-6-6 is shown at a detection wavelength of 238 nm, with a retention time of 9.096 min.

[0037] NMR measurements of compound SPUD-5-6-6 were performed using a Bruker Avance II-500 NMR spectrometer (Bruker, Switzerland). 1 H and 13 C nuclear magnetic resonance spectrum. 1 H and 13 The CNMR operating frequencies were 500 MHz and 125 MHz. Samples were dissolved in 600 μL LCD3OD at an experimental temperature of 300 K. Coupling constants (J) are expressed in Hz, chemical shifts in δ (ppm), and tetramethylsilane (TMS) was used as an internal standard. Results are as follows: Figure 2 and Figure 3 .

[0038] Table 1. NMR spectral data and C and H assignments of the compounds.

[0039]

[0040] Based on the combined NMR and HPLC data of the compound and comparison with literature, it is inferred that compound SPUD-5-6-6 is kynuric acid; it is readily soluble in methanol and chloroform, and slightly soluble in acetone. It exhibits absorption peaks at λ = 220 nm, λ = 274 nm, and λ = 302 nm. The compound's m / z = 187.8 [M-2H]+, therefore M = 189.8; its structure is as follows:

[0041]

[0042] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0043] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for extracting kynurenic acid from potato peel, characterized in that, Includes the following steps: Step 1: Freeze-dry and pulverize the potato peels to obtain potato peel powder; Step 2: Soak potato peel powder in water and extract with ultrasonic assistance. Repeat the extraction 3 times, combine the extracts, concentrate under reduced pressure to obtain crude extract SPUD. Step 3: Redissolve the crude extract SPUD in water, add ethanol, store at 4°C for 12 h, centrifuge to remove the precipitate, and add 80% ethanol twice the volume of concentrated solution. Step 4: Concentrate the supernatant to remove ethanol, redissolve in water, and separate and purify using Sephadex G-25 at a flow rate of 0.25 mL / min with water as the eluent. Collect each fraction using an automatic collector, collecting one tube every 30 min, for a total of 106 tubes. After TLC analysis, 9 components were obtained. The fractions from tubes 46-63 were combined and then concentrated under reduced pressure to obtain component SPUD-5. Step 5: After reconstitute component SPUD-5 with water, acidify it with acetic acid, add it to a cation exchange resin for adsorption, elute the adsorbed cation exchange resin with water, collect the aqueous solutions, combine them, evaporate the solvent to dryness, and obtain component SPUD-5-6; the cation exchange resin is a Dowex 50 ion exchange resin packed column. Step 6: Take component SPUD-5-6, redissolve it in water, add it to macroporous resin for adsorption, and elute the adsorbed macroporous resin with water at a flow rate of 0.5 ~ 1 mL / min. Collect the components from min 106 to 138 to obtain kynurenic acid.

2. The method as described in claim 1, characterized in that, In step 2, potato peel powder and water are added at a material-to-liquid ratio of 1:10, and ultrasonic extraction is performed for 1 hour.

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