CN112824385A - Method for preparing indirubin by taking fresh leaves of kale as raw materials - Google Patents
Method for preparing indirubin by taking fresh leaves of kale as raw materials Download PDFInfo
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- CN112824385A CN112824385A CN201911138091.4A CN201911138091A CN112824385A CN 112824385 A CN112824385 A CN 112824385A CN 201911138091 A CN201911138091 A CN 201911138091A CN 112824385 A CN112824385 A CN 112824385A
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- indirubin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
- C07D209/32—Oxygen atoms
- C07D209/36—Oxygen atoms in position 3, e.g. adrenochrome
Abstract
The invention relates to a method for preparing indirubin by taking fresh leaves of kale as a raw material, which belongs to the field of natural product extraction and comprises the following steps: (1) the method comprises the following steps of (1) soaking process, (2) indirubin synthesis, (3) indirubin extraction, (4) purification and (5) refining. The invention sets the soaking conditions as follows: temperature, adding alkali, aerating and stirring. The soaking temperature of 30 ℃ is suitable for microbial propagation, which is beneficial to the production and activity of enzyme and promotes the hydrolysis and bond breaking of the precursor substance glycoside bond of the indigo in the fresh leaves of the kalanchoe indica to form free indoxyl. The PH value of the soaked solution is adjusted to be 7, so that indoxyl is isomerized into indolone in an alkaline environment. The oxygen content in the solution is increased by aeration and stirring, the indirubin precursor is promoted to be converted into an intermediate substance for generating the indirubin, and the extraction rate and the purity of the indirubin can be improved.
Description
Technical Field
The invention belongs to the field of natural product extraction, and particularly relates to a method for preparing indirubin by taking fresh leaves of kale as a raw material.
Background
The kalanchoe (Baphicathus cusia Brem) is a perennial herb of the genus kalanchoe of the family Acanthaceae and is widely distributed in the southwest, south and east China. Ma lan Gen, jiao and Ye can be used as raw materials. The root and stem of the isatis root are used as the medicine, and the dried leaf is used as the dyers woad leaf, so that the isatis root and stem are used as the medicine, and the isatis leaf and stem are not only common clinical Chinese medicines, but also main raw materials of a plurality of Chinese patent medicines. The raw materials for preparing the indirubin mainly comprise 'blue' plants such as Indigowoad blue, isatis tinctoria and Polygonum tinctoria, wherein the content of the indirubin in fresh Indigowoad blue leaves is highest, meanwhile, the Indigowoad blue leaves and leaves contain a large amount of indirubin which is an indigo precursor substance or free indoxyl, and the finished product of the indirubin processed by the Indigowoad blue leaves and leaves has high content of the indirubin and better quality than other similar products.
Indirubin is an anti-leukemia effective component separated from natural indigo, and is a bisindole antineoplastic agent. Indirubin has inhibitory effect on various transplanted animal tumors, and can destroy leukemia cells; the indirubin has certain therapeutic effect on animal transplantation tumor and human tumor, and can cause necrotic cells with degeneration to show swelling and lytic necrosis. And the total effective rate of the indirubin for clinically treating chronic myelocytic leukemia reaches more than 90 percent, and the indirubin has the same curative effect with the clinical preferred medicament of kalimeris indica, quick curative effect and small side effect. Currently, indirubin for research in China has 3 sources: the natural product extracted from the traditional Chinese medicine dyers woad leaf and natural indigo, and the semisynthetic and fully synthesized indirubin.
The preparation method of the indirubin mainly comprises the following steps: 1> directly extracting from the raw material folium isatidis by using an organic solvent. The more fresh the horse blue is processed into indigo naturalis containing more indigo blue and indirubin, the content of the indirubin is reduced in the drying and heating extraction processes of the raw material leaves, and the indirubin precursor substances in the raw material cannot be converted into the indirubin.
2, fermenting the fresh stem leaf raw material to prepare natural indigo, and separating and extracting indirubin from the natural indigo. The traditional method for preparing indigo naturalis is, most of indigoid precursor substances are converted into indigo, and the content of indirubin in the indigo naturalis is equivalent to that in leaves.
3> semi-chemical synthesis. Adding another precursor indoloquinone for converting indirubin into fresh raw material slurry, and combining the indoloquinone with the precursor indophenol in the raw material to generate the indirubin. Or the indirubin is directly synthesized by the indigo and the indoloquinone. The reaction requires the compound indoloquinone to participate in the production.
A large number of researches show that the effective components of indigo naturalis, namely indigo and indirubin, are not completely and directly present in plants, only a small part of indigo is directly present in the plants, and stems and leaves contain a large amount of precursors of the indigo, namely indoside or free indoxyl. During the processing and soaking process of indigo naturalis, glycoside bonds in the indigo naturalis are subjected to enzymolysis and bond breaking under the microbial fermentation effect to form free indoxyl, indoxyl is isomerized into indolone under hot water or alkaline environment, and finally, the indolone of two molecules is subjected to oxidation condensation reaction under alkaline condition to form indigo blue.
The mechanism of indoxyl formation indicates that the formation of indoxyl free radicals requires oxygen from the dissolved oxygen in the original soaking solution, but oxygen is required to be supplied from the outside to the indoline diketone formed by further oxidation. Therefore, the control of system pH and the oxygen supply during enzymolysis are the key to the generation of indigo. If the indigo production is performed in the direction of indirubin, the formation of indoline dione is promoted by means of oxygen introduction, pH adjustment or the like during the enzymatic hydrolysis, and if the indigo production is performed in the direction of indigo, the oxygen content and the system pH should be controlled during the enzymatic hydrolysis to prevent the formation of indoline dione, so that the system oxygen content and pH are closely related to which indigo is formed.
By controlling the proper microorganism breeding and soaking temperature, the microorganism fermentation is accelerated, and the generation of indoxyl is promoted. The indigo precursor is converted into indirubin more by controlling the pH of the system and the oxygen supply amount.
Disclosure of Invention
Based on the defects in the background art, the invention aims to disclose a method for preparing indirubin by using fresh leaves of kale as a raw material, which improves the extraction efficiency of the indirubin.
The invention is realized by the following process steps:
a method for preparing indirubin by taking fresh leaves of kale as raw materials comprises the following steps: (1) the method comprises the following steps of (1) soaking process, (2) indirubin synthesis, (3) indirubin extraction, (4) purification and (5) refining. The method for preparing indirubin by taking the fresh leaves of kale as raw materials comprises the following specific steps:
(1) the soaking process comprises the following steps: placing fresh leaves in 30 deg.C deionized water with pH of 7, wherein the volume of water is 30 times of the fresh leaves, ventilating, stirring once for 1 hr, soaking for 48 hr, and filtering with a sieve to remove leaf residue;
(2) synthesis of indirubin: adding alkali into the filtrate to adjust the pH of the soaking solution to 10.5, heating the soaking solution to 60 ℃, simultaneously ventilating and stirring for 1h, standing and cooling;
(3) extracting indirubin: filtering the mixed soaking solution with a large filter screen to remove impurities, and filtering the filtrate with an ultrafiltration membrane to obtain crude indirubin;
(4) and (3) purification: adding 3 times of volume equivalent deionized water into the reddish brown pasty indirubin obtained in the step (3), adding hydrochloric acid to make the solution acidic, controlling the pH value to be 3-4, placing the solution in a refrigerator with the temperature set to be 2-5 ℃ for cooling and precipitation, filtering, returning the solution to be neutral to alkalescence, enabling the filtrate to pass through a macroporous resin column, firstly washing the filtrate by 6 times of water until the filtrate is nearly colorless, then washing the filtrate by 50% of ethanol, finally eluting the filtrate by 100% of acetic acid ethanol, and concentrating the eluent under reduced pressure to obtain an indirubin fine product;
(5) refining: dissolving refined indirubin in 100% ethanol under heating, rotary concentrating, and slowly cooling to obtain indirubin crystal. Washing the crystal with water, and recrystallizing with ethanol to obtain pure indirubin. Preferably, the method for preparing indirubin by using the fresh leaves of kale as raw materials comprises the following steps:
the aperture of the ultrafiltration membrane in the step (3) is 0.002 μm.
Preferably, the method for preparing indirubin by using the fresh leaves of kale as raw materials comprises the following steps: the macroporous resin filtration process in the step (4) comprises the following steps: loading the pretreated D3520 resin into a 500 x 10mm glass column, passing a sample solution through the column for adsorption, passing deionized water through the column to wash water-soluble impurities in the sample after the sample is adsorbed, passing 50% ethanol with 4BV through the column to wash fat-soluble impurities in the sample, passing the column with 100% ethanol with 6BV at a flow rate of 3BV/h to elute the sample, and collecting a sample eluent.
The invention has the advantages of
1. By setting the soaking conditions: temperature, adding alkali, aerating and stirring. The soaking temperature of 30 ℃ is suitable for microbial propagation, which is beneficial to the production and activity of enzyme and promotes the hydrolysis and bond breaking of the precursor substance glycoside bond of the indigo in the fresh leaves of the kalanchoe indica to form free indoxyl.
2. The PH value of the soaked solution is adjusted to be 7, so that indoxyl is isomerized into indolone in an alkaline environment. The oxygen content in the solution is increased by aeration and stirring, and the transformation of the indirubin intermediate substance into the indirubin precursor substance is promoted.
3. Synthesis of indirubin: the aeration and stirring increase the oxygen content in the solution and promote the generation of indirubin.
4. And (3) in the extraction process, adopting an ultrafiltration membrane combined with macroporous resin for extraction: simple operation, simplified process flow and high extraction efficiency. The ethanol used for purification and refining is safe and green, and has small toxic effect.
Detailed Description
Example 1
Influence of different oxygen amounts on crude indirubin extraction
Four portions of 1kg fresh leaves were weighed into 30 ℃ tap water having a pH of 7, wherein the volume of the water is 30 times the mass of the fresh leaves. The mixture is aerated for 1 hour and stirred once (not aerated and stirred), and after soaking for 48 hours, the mixture is filtered by a filter screen to remove leaf residues. Adding NaOH into the soaking solution to adjust the pH of the filtrate to 10.5, introducing air for 1h (without introducing air), stirring for 1h, standing, cooling, filtering the mixed soaking solution with a large filter screen to remove impurities, and filtering the filtrate with an ultrafiltration membrane with the pore diameter of 0.002 μm to obtain crude indirubin. The content of indirubin in the crude product is determined by an RP-HPLC method, and the specific data are shown in Table 1.
TABLE 1
Preferably, fermentation stirring and aeration before beating indigo. The content of indirubin in the conventional production of indigo naturalis is equivalent to that in the leaves, and the oxygen content is increased in the preparation process of indigo naturalis, so that the indirubin can be promoted to be directionally converted from the indirubin precursor.
Example 2
Effect of pH on indirubin
Weighing 3 parts of 1kg of fresh leaves, and placing the fresh leaves in tap water with the pH of 3.5, 5.5 and 7 at 30 ℃, wherein the volume of the tap water is 30 times of the mass of the fresh leaves. Ventilating and stirring once for 1 hour, soaking for 48 hours, and filtering by a filter screen to remove leaf residues. Adding NaOH into the soaking solution to adjust the pH of the filtrate to 10.5, ventilating and stirring for 1h, standing and cooling, filtering the mixed soaking solution with a large filter screen to remove impurities, filtering the filtrate with an ultrafiltration membrane with the pore diameter of 0.002 mu m to obtain indirubin paste, and respectively weighing the paste, which is shown in the following table.
TABLE 2
| Immersion pH | Indirubin g/kg |
| 3.5 | 4.82 |
| 5.5 | 6.04 |
| 7 | 6.21 |
Example 3
Using ultrafiltration membranes
1kg of fresh leaves was weighed into 30 ℃ tap water having a pH of 7, wherein the volume of the water was 30 times the mass of the fresh leaves. Ventilating and stirring once for 1 hour, soaking for 48 hours, and filtering by a filter screen to remove leaf residues. Adding NaOH into the soak solution to adjust the pH value of the filtrate to 10.5, then introducing air for 1h, introducing air and stirring for 1h, standing and cooling, filtering the mixed soak solution by a large filter screen to remove impurities, and filtering the filtrate by an ultrafiltration membrane with the pore diameter of 0.002 mu m to obtain crude indirubin, wherein the crude indirubin is 54.25g, the content of the indirubin is 11.13 percent, and the yield of the indirubin is 97 percent.
Example 4
Conventional processes without ultrafiltration membranes
1kg of fresh leaves was weighed into 30 ℃ tap water having a pH of 7, wherein the volume of the water was 30 times the mass of the fresh leaves. Ventilating and stirring once for 1 hour, soaking for 48 hours, and filtering by a filter screen to remove leaf residues. Adding NaOH into the soaking solution to adjust the pH of the filtrate to 10.5, introducing air for 1h, adding 4% fresh lime milk, stirring and beating indigo for 40min, filtering to obtain precipitate, filtering the filtrate again for recovery, adding water into the precipitate, mixing, and removing impurities with water to obtain crude indigo. Adding 4 times of ethanol into the indigo paste, stirring for 300W, performing ultrasonic treatment for 40min, filtering, extracting the filter residue with ethanol twice, mixing the filtrates, rotary evaporating, concentrating, recovering ethanol solvent, and adding distilled water with the same amount as ethanol. Concentrating and recovering 1/2 water, cooling, and filtering to obtain filter residue which is crude indirubin product 55.32g, the content of indirubin is 8.68%, and the yield of indirubin is 89%.
Compared with the prior production process, the ultrafiltration membrane filtration is simple and has high efficiency.
Example 5
Purification of indirubin
1kg of fresh leaves was weighed into 30 ℃ tap water having a pH of 7, wherein the volume of the water was 30 times the mass of the fresh leaves. Ventilating and stirring once for 1 hour, soaking for 48 hours, and filtering by a filter screen to remove leaf residues. Adding NaOH into the soak solution to adjust the pH value of the filtrate to 10.5, introducing air for 1h, introducing air and stirring for 1h, standing and cooling, filtering the mixed soak solution with a large filter screen to remove impurities, and filtering the filtrate with an ultrafiltration membrane with the pore diameter of 0.002 mu m to obtain the crude indirubin. 5g of the pretreated D3520 resin were loaded onto a 500X 10mm glass column BV of 30 ml. Dissolving 1kg of crude indigo indirubin prepared from fresh leaves of kale in ethanol to prepare a sample solution, and flowing 3BV of the sample solution into a chromatographic column at the flow rate of 1BV/h for adsorption treatment. And after adsorption, washing water-soluble impurities by using deionized water, washing fat-soluble impurities by using 4BV 50% ethanol, finally dissociating by using 100% ethanol with the amount of 6BV at the flow rate of 3BV/h, collecting eluent, and performing rotary concentration to obtain 48.47g of wet indigo indirubin product.
Dissolving the obtained indirubin paste with 80% ethanol under heating, concentrating at low temperature to obtain reddish brown crystal layer, filtering to remove water and wash crystal, recovering filtrate, and recrystallizing with 80% ethanol for 3 times to obtain indirubin crystal. The purity of the obtained indirubin is higher than 97.3 percent, and the recovery rate of the indirubin reaches 78 percent.
The purification process only uses non-toxic ethanol as a solvent, and does not use solvents such as methanol, dichloromethane, chloroform and the like in the prior art, so that the finished product is green and safe.
Claims (4)
1. A method for preparing indirubin by taking fresh leaves of kale as raw materials is characterized by comprising the following steps: (1) the method comprises the following steps of (1) soaking process, (2) indirubin synthesis, (3) indirubin extraction, (4) purification and (5) refining.
2. The method for preparing indirubin by using fresh leaves of kale as raw materials according to claim 1, which comprises the following steps:
(1) the soaking process comprises the following steps: placing fresh leaves in 30 deg.C deionized water with pH of 7, wherein the volume of water is 30 times of the fresh leaves, ventilating, stirring once for 1 hr, soaking for 48 hr, and filtering with a sieve to remove leaf residue;
(2) synthesis of indirubin: adding alkali into the filtrate to adjust the pH of the soaking solution to 10.5, heating the soaking solution to 60 ℃, simultaneously ventilating and stirring for 1h, standing and cooling;
(3) extracting indirubin: filtering the mixed soaking solution with a large filter screen to remove impurities, and filtering the filtrate with an ultrafiltration membrane to obtain crude indirubin;
(4) and (3) purification: adding 3 times of volume equivalent deionized water into the reddish brown pasty indirubin obtained in the step (3), adding hydrochloric acid to make the solution acidic, controlling the pH value to be 3-4, placing the solution in a refrigerator with the temperature set to be 2-5 ℃ for cooling and precipitation, filtering, returning the solution to be neutral to alkalescence, enabling the filtrate to pass through a macroporous resin column, firstly washing the filtrate by 6 times of water until the filtrate is nearly colorless, then washing the filtrate by 50% of ethanol, finally eluting the filtrate by 100% of ethanol, and concentrating the eluent under reduced pressure to obtain an indirubin fine product;
(5) refining: dissolving refined indirubin in 100% ethanol under heating, rotary concentrating, and slowly cooling to obtain indirubin crystal. Washing the crystal with water, and recrystallizing with ethanol to obtain pure indirubin.
3. The method for preparing indirubin from fresh leaves of kale as claimed in claim 2, wherein:
the aperture of the ultrafiltration membrane in the step (3) is 0.002 μm.
4. The method for preparing indirubin from fresh leaves of kale as claimed in claim 2, wherein: the macroporous resin filtration process in the step (4) comprises the following steps: loading the pretreated D3520 resin into a 500 x 10mm glass column, passing a sample solution through the column for adsorption, passing deionized water through the column to wash water-soluble impurities in the sample after the sample is adsorbed, passing 50% ethanol with the amount of 4BV through the column to wash fat-soluble impurities in the sample, passing the column at the flow rate of 3BV/h with 100% ethanol with the amount of 6BV to elute the sample, and collecting a sample eluent.
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2019
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Application publication date: 20210521 |