CN115177646B - Method for regulating and controlling acidified crude extract of bluish dogbane and extracting bluish alkaloids in grading manner - Google Patents
Method for regulating and controlling acidified crude extract of bluish dogbane and extracting bluish alkaloids in grading manner Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
The invention discloses a method for conducting, regulating and controlling an acidified crude extract of Kalimeris indica and extracting a Kalimeris indica alkaloid in a grading way, and particularly relates to the field of biological medicines, comprising the following steps of: s1, preparing a crude extract suspension of the bluish red: adding 2-10 times of water into Ma Landian crude extract for dilution, stirring for 0.1-1 hour, wherein the stirring speed is 100-1000r/min, regulating the electric conductivity of filtrate to 8000-1.2 ten thousand (mu s/cm), centrifugally filtering, and adding 2-5 times of water into filter residues for dilution to obtain a suspension of the crude extract of the Indian kalimeris herb. The invention adopts conductivity to control the concentration of ions in the crude extract suspension of the bluish dogbane and the acidification reaction process, controls the conductivity of the suspension to 8000-1.2 ten thousand, and can remove impurities such as free calcium hydroxide, silicate, mud dust and the like; the conductance of the acidified filtrate is regulated to be 6-10 ten thousand (mu s/cm), and calcium carbonate particles can be completely and uniformly acidified to release water-insoluble alkaloids such as indigo, indirubin and the like; and controlling the ion concentration in acidification and water washing through conductivity, and completely removing impurities to obtain the refined powder of the crude extract of the bluish dogbane.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for conducting, regulating and controlling an acidified crude extract of bluish dogbane and extracting bluish dogbane alkaloid in a grading way.
Background
The root, stem and leaf of the herb of Ardisia japonica is called as the root of the herb of Buddha's shrub like perennial herb of Acanthaceae, and commonly called as the indigo plant. The main medicinal components of the indigo naturalis in the Chinese angelica-longhui pills are indigo and indirubin, which are traditional medical medicines with long history in China, are genuine Chinese medicinal materials and are recorded in Chinese pharmacopoeia, and are used for treating chronic granulocytic leukemia, anti-inflammatory and virus and the like. The natural indigo does not contain toxic components such as aniline in the synthetic indigo, is irreplaceable for the synthetic indigo, and is an important variety in the food additive-blue pigment allowed by China.
The blue is widely planted in southwest poor mountain areas such as Guizhou, yunnan and the like, the current area is more than 50 ten thousand mu, the local minority nationality such as Miao nationality, shui nationality, dong nationality, yao nationality, buyi nationality and the like is prepared into indigo paste through the traditional process, the blue becomes the main natural dye of Miao nationality wax printing technology-indigo inheritance thousands of years, and the blue is listed into national non-material cultural heritage in 2006, and the regional printing and dyeing raw materials are radiated nationally or even worldwide.
The folium Isatidis contains indigo, indirubin, tryptanthrin, quinazolinone, etc. alkaloids, sterols, polysaccharides, organic phenols, etc. The alkaloid is an important active ingredient in the stem and leaf of the indigowoad leaf, is used for processing the indico powder, the indico extract, the indigotin extract and the indirubin extract, and is applied to dyes, cosmetics, food pigments and medicines.
The indigo crude extract comprises indigo paste, indigo coarse powder and indigo powder. The domestic workshop is processed by adopting the traditional process, the cut stems and leaves are soaked in cement pool water for 3-7 d, the stems and leaves are rotten, the stems, the branches and the leaves are removed, lime is added for flower beating, standing and precipitation are carried out, the indigo paste is obtained by filtration, and the indigo coarse powder or the indigo powder is produced by sun-drying and crushing. The traditional process has low extraction rate, the content of inorganic matters such as calcium, silicate and the like and sediment is up to more than 90 percent, the content of active ingredients such as indigo indirubin and the like is less than 10 percent, the content of impurities is high, and the industrialized preparation technology for separating and purifying the crude extract of the indigo paste is lacking at present.
Duan Xiaoying and the like discuss four influencing factors of indigo ethanol extraction by taking the indirubin extraction rate as an index, and the indirubin extraction rate can reach about 77 percent. Huang Hui and the like respectively optimize factors such as extraction solvents (acetonitrile, ethanol, ethyl acetate, chloroform, methanol, dimethylformamide), extraction temperature, extraction time, material ratio and the like which influence the natural indigo heating reflux extraction method, the ultrasonic extraction method and the microwave extraction method, and take the number of common peaks of a UPLC (ultra high performance liquid chromatography), the total peak area of characteristic fingerprint peaks and the content of indirubin as evaluation indexes. Zhang Qingsong and the like have studied the extraction of indirubin in indigo by different solvents (ethyl acetate, acetone, chloroform and 75% ethanol), compared the solubility of indirubin in different solvents, and reported the photodegradation of indirubin in methylene chloride to produce indirubin, isatoic anhydride, tryptanthrin, anthranilic acid and tryptophan. Chen Ying and the like are used for researching the chloroform leaching condition of extracting natural indigo with different particle sizes. The above documents only study the extraction of indirubin in natural indigo, and have the problems of low separation extraction rate, more solvents, high cost and the like, and the related technologies for comprehensively separating, purifying and controlling degradation of active substances with high content of indirubin, indigo, tryptanthrin, polyphenol and the like are lacking.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks of the prior art, embodiments of the present invention provide a method for conducting, regulating and controlling an acidified crude extract of kalimeris indica and extracting a kalimeris indica alkaloid in a graded manner, so as to solve the problems set forth in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: the method for conducting, regulating and controlling the acidification of the crude extract of the bluish with electric conduction and extracting the bluish alkaloid in a grading way comprises the following steps:
s1, preparing a crude extract suspension of the bluish red: adding 2-10 times of water into Ma Landian crude extract for dilution, stirring for 0.1-1 hour at the stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to 8000-1.2 ten thousand (mu s/cm), centrifugally filtering, and adding 2-5 times of water into filter residues for dilution to obtain a bluish crude extract suspension;
s2, acidifying a crude extract suspension of the bluish red: adding 10% -25% hydrochloric acid solution into the crude extract suspension of the bluish, acidifying at 40-80 ℃, stirring for 0.5-2 hours at a stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to be 6-10 ten thousand (mu s/cm), adding 2-5 times of hot water into filter residues, stirring and cleaning for 1-5 times, regulating the electric conductivity of the filtrate to be 1000-5000 (mu s/cm), filtering to obtain acidified filter residues, drying, grinding into powder with an average particle size of 20-180 mu m, and obtaining refined powder of the bluish crude extract;
s3, preparing indirubin extract: adding an alcohol-soluble solvent into the refined powder of the crude extract of the bluish in the step S2 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying filter residues, merging extract liquid, then carrying out low-temperature vacuum recovery of the solvent at the dark, adsorbing concentrate by 5-15 times of polar medium, adding 10-20 times of polar solvent into the concentrate, washing for 1-5 times, filtering, merging washing liquid, and carrying out low-temperature vacuum recovery of the solvent to obtain the indirubin extract with the indirubin content of 10% -45%;
s4, preparing an indigo extract: adding a medium-polarity solvent into the filter residue dried in the step S3 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times under the condition of avoiding light, carrying out centrifugal filtration, drying the filter residue, merging the extract solutions, carrying out low-temperature vacuum recovery of the solvent under the condition of avoiding light, and drying the concentrate into powder to obtain an indigo extract with the indigo content of 20% -60%;
s5, preparing a bluish amine ketone extract: adding a polar solvent into the filter residue in the step S4 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying the filter residue, merging the extract liquid, carrying out vacuum recovery of the solvent under the dark condition, and drying the concentrate into powder to obtain the bluish amine ketone extract with the tryptanthrin content of more than 15%.
Further, the crude extract of the indigotin is indigotin dry powder or natural indigo powder prepared by the traditional fermentation process of the stem and leaf of the indigotin, wherein the content of the indigotin is 0.5-7%, the indigotin is 0.1-0.5% and the tryptanthrin is 0.1-0.2%.
Further, the alcohol-soluble solvent is one of methanol, ethanol and propanol or 60-95% alcohol-water solvent.
Further, the medium polarity solvent is one or a mixed solvent of ethyl acetate, chloroform, dichloromethane and acetone.
Further, the polar solvent is one or a mixed solvent of DMF and DMSO.
Further, the ultrasonic wave adopts 50hz, and the proportion of ultrasonic power to the crude extract of the bluish is 100-800W: 5-50 g.
Furthermore, the light shielding is to isolate the solvent system of the bluish active substance from light rays, including sunlight, LED light, UV and the like, by adopting dark vessels or cloths such as black, brown and the like.
Further, the low-temperature vacuum is at a temperature of 50-60 ℃ and a vacuum degree of 500-760 mmHg.
Further, the polar medium is silica gel, alumina, diatomite, macroporous resin, etc.
The invention has the technical effects and advantages that:
1. the invention adopts conductivity to control the concentration of ions in the crude extract suspension of the bluish dogbane and the acidification reaction process, controls the conductivity of the suspension to 8000-1.2 ten thousand, and can remove impurities such as free calcium hydroxide, silicate, mud dust and the like; the conductance of the acidified filtrate is regulated to be 6-10 ten thousand (mu s/cm), and calcium carbonate particles can be completely and uniformly acidified to release water-insoluble alkaloids such as indigo, indirubin and the like; the ion concentration in acidification and water washing is controlled through conductivity, impurities are completely removed, and the product appearance is sky blue fluffy powder, wherein the indigo is more than 20%, the indirubin is more than 0.5%, and the product is obtained by the traditional acid method.
2. Aiming at the problem that alkaloids such as indigo and indirubin are extremely easy to degrade in the soluble neutralization and visible light, a solvent system of a bluish active substance is isolated from light rays by adopting dark vessels or cloths such as black, brown and the like, including sunlight, LED light, UV and the like, and then the bluish active substance is subjected to fractional ultrasonic extraction by different polar solvents, so that alkaloids such as high-content indirubin, indigo, tryptanthrin and the like are sequentially obtained, the fractional enrichment of alkaloids in a bluish crude extract is realized, the degradation of the traditional solvent extracted alkaloids is avoided, and the extraction rate is higher than 95%.
Drawings
FIG. 1 is an HPLC chart of a Kalimeris leaf in the present invention.
FIG. 2 is a graph showing the degradation of 254nm indigo light in the present invention.
FIG. 3 is a graph showing the degradation of 365nm indigo light in the present invention.
FIG. 4 is a graph showing the photodegradation of 254nm indirubin in the present invention.
FIG. 5 is a graph showing the degradation of 365nm indirubin light in the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a method for conducting, regulating and controlling an acidified crude extract of Kalimeris indica and extracting a Kalimeris indica alkaloid in a grading manner, which is characterized by comprising the following steps of:
s1, preparing a crude extract suspension of the bluish red: adding 2-10 times of water into Ma Landian crude extract for dilution, stirring for 0.1-1 hour at the stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to 8000-1.2 ten thousand (mu s/cm), centrifugally filtering, and adding 2-5 times of water into filter residues for dilution to obtain a bluish crude extract suspension;
s2, acidifying a crude extract suspension of the bluish red: adding 10% -25% hydrochloric acid solution into the crude extract suspension of the bluish, acidifying at 40-80 ℃, stirring for 0.5-2 hours at a stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to be 6-10 ten thousand (mu s/cm), adding 2-5 times of hot water into filter residues, stirring and cleaning for 1-5 times, regulating the electric conductivity of the filtrate to be 1000-5000 (mu s/cm), filtering to obtain acidified filter residues, drying, grinding into powder with an average particle size of 20-180 mu m, and obtaining refined powder of the bluish crude extract;
s3, preparing indirubin extract: adding an alcohol-soluble solvent into the refined powder of the crude extract of the bluish in the step S2 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying filter residues, merging extract liquid, then carrying out low-temperature vacuum recovery of the solvent at the dark, adsorbing concentrate by 5-15 times of polar medium, adding 10-20 times of polar solvent into the concentrate, washing for 1-5 times, filtering, merging washing liquid, and carrying out low-temperature vacuum recovery of the solvent to obtain the indirubin extract with the indirubin content of 10% -45%;
s4, preparing an indigo extract: adding a medium-polarity solvent into the filter residue dried in the step S3 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times under the condition of avoiding light, carrying out centrifugal filtration, drying the filter residue, merging the extract solutions, carrying out low-temperature vacuum recovery of the solvent under the condition of avoiding light, and drying the concentrate into powder to obtain an indigo extract with the indigo content of 20% -60%;
s5, preparing a bluish amine ketone extract: adding a polar solvent into the filter residue in the step S4 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying the filter residue, merging the extract liquid, carrying out vacuum recovery of the solvent under the dark condition, and drying the concentrate into powder to obtain the bluish amine ketone extract with the tryptanthrin content of more than 15%.
In a preferred embodiment, the crude extract of the indigotin is indigotin dry powder or indigo powder prepared by the traditional fermentation process of the stem and leaf of the indigotin, wherein the content of the indigotin is 0.5-7%, the indigotin is 0.1-0.5% and the tryptanthrin is 0.1-0.2%.
In a preferred embodiment, the alcohol-soluble solvent is one of methanol, ethanol, propanol or 60% -95% alcohol-water solvent.
In a preferred embodiment, the medium polarity solvent is one or a mixture of ethyl acetate, chloroform, methylene chloride, acetone.
In a preferred embodiment, the polar solvent is one or a mixture of DMF, DMSO.
In a preferred embodiment, 50hz is used for the ultrasound, and the ratio of the ultrasound power to the crude extract of the bluish is 100 to 800W: 5-50 g.
In a preferred embodiment, light protection is to isolate the bluish active solvent system from light, including sunlight, LED light, UV, etc., using dark vessels or cloths of black, brown, etc.
The crude extract of the bluish is prepared by controlling calcium, silicate and other inorganic matters and sediment in suspension by adopting conductivity, diluting with 2-10 times of water, preferably 4-6 times of water, stirring for 0.1-1 hour, stirring at a speed of 100-1000r/min, centrifugally filtering, regulating the conductivity of filtrate to 8000-1.2 ten thousand, and removing free calcium hydroxide, silicate, mud dust and other impurities.
In order to effectively and uniformly release water-insoluble alkaloids such as indigo, indirubin and the like from calcium carbonate particles in the acidification reaction process, the invention optimizes the concentration of hydrochloric acid, the reaction temperature and the reaction time, and has the advantages of low concentration of hydrochloric acid solution below 10%, large volume, long acidification time, incomplete calcium carbonate particle reaction and low alkaloid content in an acidified product; the concentration of hydrochloric acid solution above 25% is high, heat release, fuming and reaction foam are serious, foam coated calcium carbonate particles float upwards and are separated from acid to cause non-uniform and complete reaction, the alkaloid content in an acidified product is unstable, therefore, the invention adopts 10% -25% hydrochloric acid solution to be added into a crude extract suspension of the indian kalium, the acidification temperature is 40-80 ℃, the stirring speed is preferably 300-500 r/min, stirring is carried out for 1.5 hours, the conductivity of filtrate is regulated to be 6-10 ten thousand (mu s/cm), preferably 8 ten thousand mu s/cm, the calcium carbonate particles can be completely and uniformly acidified, water-insoluble alkaloids such as indigotin blue and indirubin are released, the filter residue is stirred and washed for 3 times by adding hot water 2-5 times, the conductivity of filtrate is regulated to be 1000-5000 (mu s/cm), filtering is carried out, the ion concentration in the acidification and water washing processes can be simply, conveniently and accurately known through conductivity control, the acidification and washing process is carried out, the obtained filter residue is dried to obtain a crude extract fine powder of the indigotten blue, the product has the appearance of sky blue powder, wherein indigotten blue is more than 20%, indigotin the indigotin and indigotin is more than 3%.
In order to enrich high-content indigotin active matters, the existing literature adopts organic solvent extraction including modes of ultrasonic extraction, microwave extraction and the like, researches on the influence of the types of organic solvents, the particle sizes and the light intensities of fine powder of the indigotin crude extract on the extraction rate, the degradation speed and the content of indigotin, but does not consider the degradation of the indigotin and indigotin by solvents and light in the extraction process, firstly, the fine powder of the indigotin crude extract is ground into powder with the average particle size of 20-180 mu m, then the organic solvent is used for ultrasonic fractional extraction under the dark place, so that the indigotin active matters rich in indigotin and indigotin are obtained, and the indigotin content of the indigotin is more than 95% of filter residues because the indigotin is easily dissolved in polar alcohol solvents such as acetone, methanol, ethanol and the like, and the solubility of indigotin the solvents are small, and the solubility of indigotin the medium polarity such as ethyl acetate, chloroform and the like; extracting indigo with medium polar solvent of ethyl acetate or chloroform or mixed solvent, wherein the content of indigo in the indigo extract is 20% -60%, extracting alkaloid such as azulene and quinazolinone with polar solvent of DMF or DMSO, and grading and enriching alkaloid in crude extract.
The ultrasonic wave has good penetrability, is favorable for solvent permeation and quick disintegration of the crude extract fine powder of the bluish with the average grain diameter of 20-180 mu m, and the ultrasonic wave adopts 50hz, and the proportion of the ultrasonic power and the crude extract of the bluish is 100-800W: 5-50 g, preferably 400w:20g, solvent volume to solid material ratio of 4:1 to 15:1, carrying out ultrasonic reflux extraction for 1-5 times, preferably 2-3 times, at 30-70 ℃. Most solvents of the fractional extraction are recovered by low-temperature vacuum concentration, the temperature is 50-60 ℃, the vacuum degree is 500-760 mHg, and the solvent loss is lower than 2%. The separation principle of silica gel is to separate substances according to different adsorption forces on silica gel, and generally, substances with larger polarity are easily adsorbed by silica gel, substances with weaker polarity are not easily adsorbed by silica gel, and the whole chromatography process is adsorption, desorption, re-adsorption and re-desorption processes. Wherein, the micro-holes have different adsorption capacities to different compounds, and then the proper eluent is selected for elution so as to achieve separation.
Example 1: HPLC analysis of the Malay alkaloid
1. Standard solution preparation
Weighing indirubin standard (mass fraction is more than or equal to 97%) 1.00mg to 100mL volumetric flask, adding N, N-dimethylformamide (DMF chromatographic purity), and ultrasonically dissolving for 30 minutes to prepare reference substance solution with mass concentration of 100 mug/mL. 0.4mL, 0.8mL, 1.2mL, 1.6mL and 2.0mL of stock solution are respectively measured into 25mL volumetric flasks by using a pipette, DMF is fixed in volume, and samples are taken for liquid phase analysis after shaking evenly.
Weighing an indigo standard (the mass fraction is more than or equal to 98%) into a volumetric flask of 10.10mg to 100ml, adding N, N-dimethylformamide (DMF chromatographic purity), and performing ultrasonic dissolution for 30 minutes. After dissolution, the solution is diluted in a gradient manner, 0.4mL, 0.8mL, 1.2mL, 1.6mL and 2.0mL of stock solution are respectively measured into 25mL volumetric flasks by a pipette, DMF is used for constant volume, and sampling is carried out after shaking evenly for liquid phase analysis.
The tryptanthrin standard (the mass fraction is more than or equal to 98%) is weighed into a volumetric flask with the volume of 1.00mg to 100mL, N-dimethylformamide (DMF chromatographic purity) is added, and the mixture is dissolved for 30 minutes by ultrasonic, so as to prepare a reference substance solution with the mass concentration of 100 mug/mL. After dissolution, the solution was diluted in a gradient manner, and 0.4mL, 0.8mL, respectively, were measured with a pipette,
1.2mL, 1.6mL and 2.0mL stock solutions are put into a 25mL volumetric flask, DMF is used for constant volume, and sampling is carried out after shaking evenly for liquid phase analysis.
2. Preparation of test samples
Weighing 5mg of crude extract of herba Kalimeridis or indigo naturalis powder, placing in 100mL volumetric flask, adding dimethyl sulfoxide 90mL, ultrasound for 30Min, cooling, adding DMSO to scale, and filtering with 0.22 μm filter membrane. Liquid phase analysis was performed.
Weighing 10g of dried leaves of Kalimeris indica (dried at 60 ℃ and with the water content of 5.6%), carrying out heat extraction for 2h by 200mL of chloroform, extracting for 2 times, combining the extracting solutions, concentrating and recovering the solvent, adding 10mL of DMSO into the concentrate, carrying out ultrasonic treatment for 30Min, cooling, adding DMSO into the concentrate, fixing the volume to a scale, and filtering by a 0.22 mu m filter membrane. Liquid phase analysis was performed.
HPLC conditions
Color column YMC-AC-ODSA (250 mm. Times.4.6 mm,5 μm), mobile phase methanol: water=70:30, detection wavelength 290nm. The flow rate is 1mL/min, the column temperature is 30 ℃, and the sample injection amount is 20 mu L.
TABLE 1 indirubin, indigo and tryptanthrin content in the leaves and crude extract of Kalimeris indica
Example 2: electrical conductance regulating and optimizing extraction of bluish alkaloid
A. Influence of different temperatures on indigo content in crude extract powder of acidified product of Kalimeris indica
3 parts of crude extract X-1 of Kalimeris indica (indigo content 6.22%) were taken in 30g, respectively. 300ml of water was added and homogenized by stirring in a water bath at 70, 80 and 90℃for 10 minutes, respectively, and 50ml of 20% HCl were slowly added dropwise, respectively. Reaction 45min, ph=around 6. The upper liquid was centrifuged to give a conductance of 98.9, 103.5 and 130.1ms/cm, respectively. Washing with boiled water three times, 300ml each time. The conductance after washing was 0.609, 0.657 and 0.616ms/cm, respectively. After washing, the mixture was centrifuged, and the solids were dried to S-2, S-1 and S-3, respectively. Taking 10mg of acidified crude extract powder of the Indian kalimeris herb, fixing the volume of the crude extract powder to a 100ml volumetric flask by dimethyl sulfoxide, and quantitatively analyzing the indigotin crude extract, the refined extract powder of the Indian kalimeris herb, the indirubin extract and the indigotin content of the indigotin extract by high performance liquid chromatography according to the method described in the 2 nd item of the 'example 1'. The data are shown in table 2.
B. Influence of different acid addition amounts on indigo content in the acidified product, namely, the crude extract powder of the bluish red
Three portions of crude extract X-1 (indigo content 6.22%) were taken, 30g each. 300ml of water was added and homogenized in a water bath at 80℃for 10 minutes with slow dropwise addition of 50ml, 60ml, 70ml of 20% HCl, respectively. The reaction is carried out for 45min, and the pH is about 6, 5 and 2-3 respectively. The upper liquid was centrifuged to give a conductance of 103.5, 115.5 and 114.16ms/cm, respectively. Washing with boiled water three times, 300ml each time. The conductance after washing was 0.657, 0.732 and 0.497ms/cm, respectively. After washing, the mixture was centrifuged, and the solids were dried to S-1, S-4 and S-5, respectively. Taking 10mg of acidified crude extract powder of the Indian kalimeris herb, fixing the volume of the crude extract powder to a 100ml volumetric flask by dimethyl sulfoxide, and quantitatively analyzing the indigotin crude extract, the refined extract powder of the Indian kalimeris herb, the indirubin extract and the indigotin content of the indigotin extract by high performance liquid chromatography according to the 2 nd item of the 'example 1', and measuring the content of the indigotin by the method. The data are shown in table 2.
C. Influence of different feed liquid ratios on indigo content in acidified product, namely, crude extract powder of Kalimeris indica
Three crude extracts of Kalimeris indica X-1 (indigo content 6.22%) were taken at 30g. 300ml, 450ml and 600ml of water are added respectively, stirred in a water bath at 80 ℃ for 10 minutes and homogenized, and 50ml of 20% HCl is slowly added respectively. The reaction was carried out for 45min. The upper liquid was centrifuged to give conductances of 114.16, 78.38 and 65.6ms/cm, respectively. Washing with boiled water three times, 300ml each time. The conductance after washing was 0.497, 0.677, 0.556ms/cm, respectively. After washing, the mixture was centrifuged, and the solids were dried to S-5, S-7 and S-9, respectively. Taking 10mg of acidified crude extract powder of the Indian kalimeris herb, fixing the volume of the crude extract powder to a 100ml volumetric flask by dimethyl sulfoxide, and quantitatively analyzing the indigotin crude extract, the refined extract powder of the Indian kalimeris herb, the indirubin extract and the indigotin content of the indigotin extract by high performance liquid chromatography according to the 2 nd item of the 'example 1', and measuring the content of the indigotin by the method. The data are shown in table 2. D. Influence of different reaction times on the indigo content in the acidified product, the crude extract powder of blua.
Three crude extracts of Kalimeris indica X-1 (indigo content 6.22%) were taken at 30g. 450ml of water are added, stirred in a water bath at 80 ℃ for 10 minutes and homogenized, 70ml of 20% HCl are respectively slowly added dropwise. The reactions were carried out for 30, 45 and 60min, respectively. The upper liquid conducions were 86.5, 78.38, 104.5ms/cm, respectively. Washing with boiled water three times, 300ml each time. The conductance after washing was 0.395, 0.677 and 0.396ms/cm, respectively. After washing, the mixture was centrifuged, and the solids were dried to S-11, S-7 and S-12, respectively. Taking 10mg of acidified crude extract powder of the Indian kalimeris herb, fixing the volume of the crude extract powder to a 100ml volumetric flask by dimethyl sulfoxide, and quantitatively analyzing the indigotin crude extract, the refined extract powder of the Indian kalimeris herb, the indirubin extract and the indigotin content of the indigotin extract by high performance liquid chromatography according to the 2 nd item of the 'example 1', and measuring the content of the indigotin by the method. The data are shown in table 2.
Results: table 2 shows the results of quantitative analysis of the indigo content of the refined powder of crude extract of Kalimeris indica under different acidification conditions. As can be seen from table 1, the optimal process conditions for acidification of crude extract of kalimeris are: the feed liquid ratio is 1: 70ml of 20% HC were slowly added dropwise after homogenization in a water bath at 80℃for 10 min. The reaction was carried out for 45min. The indigo content in the product of the crude extract refined powder of the bluish after acidification can reach about 40 percent.
TABLE 2 influence of different acidification conditions on the indigo content in the crude extract powder of Kalimeris indica
Example 3: preparation of refined powder of crude extract of herba Kalimeridis
(1) Preparation of crude extract suspension of herba Kalimeridis
Adding 2-10 times of water into Ma Landian crude extract for dilution, stirring for 0.1-1 hr at stirring speed of 100-1000r/min, regulating filtrate conductivity to 8000-1.2 ten thousand (μs/cm), centrifuging, filtering, and adding 2-5 times of water into filter residue for dilution to obtain herba Kalimeridis crude extract suspension.
(2) Acidification of crude extract suspension of Kalimeris indica
Adding 10% -25% hydrochloric acid solution into the crude extract suspension of the bluish, acidifying at 40-80 ℃, stirring for 0.5-2 hours at a stirring speed of 100-1000r/min, regulating the electric conductivity of the filtrate to be 6-10 ten thousand (mu s/cm), adding 2-5 times of hot water into the filter residue, stirring and cleaning for 1-5 times, regulating the electric conductivity of the filtrate to be 1000-5000 (mu s/cm), filtering to obtain acidified filter residue, drying, grinding into powder with an average particle size of 20-180 mu m, and obtaining the refined powder of the bluish crude extract.
Example 4: photodegradation of indigo and indirubin in organic solvents
The experimental method comprises the following steps:
A. photodegradation of indigo in N, N-dimethylformamide
An indigo solution with a concentration of 0.005,0.01mg/ml was irradiated with ultraviolet light having a wavelength of 254nm, and a cuvette of 1cm was used as a photoreaction vessel. And scanning the indigo solution in a wave band of 230-800nm by taking DMF as a blank control on an ultraviolet-spectrophotometer, and researching the influence of light on degradation of the indigo in the DMF solution by taking absorbance of the indigo solution at 610nm at different concentrations as parameters. It can be seen from FIG. 3 that the indigo solutions of both concentrations degrade faster in 0-10min under light of 254nm wavelength. The higher the concentration, the faster the degradation.
An indigo solution with a concentration of 0.005,0.01mg/ml was irradiated with ultraviolet light having a wavelength of 365nm, and a cuvette of 1cm was used as a photoreaction vessel. The indigo solution was scanned in the wavelength band of 230-800nm on an ultraviolet-spectrophotometer with DMF as a blank, and the effect of light on the degradation of indigo in DMF solution was studied based on absorbance at 610nm as a parameter. The degradation rate of indigo at 254nm is much faster than at 365nm, as evident by comparing the degradation rates at 365nm and 254 nm. And the greater the concentration, the longer the time required for complete degradation.
B. Photodegradation of indirubin in ethyl acetate
The indirubin solution with the ultraviolet light irradiation concentration of 0.003 and 0.006mg/ml and the wavelength of 254nm is adopted, the solvent is ethyl acetate solvent, and the cuvette with the wavelength of 1cm is adopted as a photoreaction container. The effect of light on the degradation of indirubin in ethyl acetate solution was studied on an ultraviolet-spectrophotometer with ethyl acetate as a blank control and according to absorbance at 531nm as a parameter. As can be seen from fig. 3, under the irradiation of 254nm wavelength, the two concentrations of indigo solution degrade faster in 0-3min, and the higher the concentration, the faster the degradation, indicating that indirubin degrades photo-fast at 254 nm.
The indirubin solution with the concentration of 0.005 and 0.01mg/ml is irradiated by ultraviolet light with the wavelength of 365nm, and a cuvette with the concentration of 1cm is used as a photoreaction container. The effect of light on the degradation of indirubin in ethyl acetate solution was studied on an ultraviolet-spectrophotometer with ethyl acetate as a blank control and according to absorbance at 531nm as a parameter. The photodegradation rate at 365nm and 254nm is obviously shown, and the photodegradation rate of indirubin at 254nm is far faster than that at 365 nm. And the greater the concentration, the longer the time required for complete degradation. Indirubin undergoes little degradation at 365nm for two hours. The absorbance thereof may be somewhat floating because the solvent ethyl acetate is somewhat volatilized in the photodegradation reaction.
Because the indigo indirubin has the problem of light drop in an organic solvent, the invention adopts light-shading treatment in the extraction process.
Example 5: influence of different solvents on indigo and indirubin content in crude extract powder of Kalimeris indica
Four parts of refined powder of crude extract of Kalimeris indica are taken to obtain 5.0g. 50ml of ethanol, methanol, ethyl acetate, and dichloromethane were added, respectively. Ultrasonic extraction is carried out for 30min. Filtering the extractive solution, adding 50ml solvent respectively, extracting twice, mixing the two extractive solutions, and spin drying. Respectively obtaining four samples after ultrasonic extraction of the solvent and extracting slag. Taking 5mg of the extracted samples and 10mg of the extracted residues respectively, fixing the volume of the extracted residues to a 100ml volumetric flask by dimethyl sulfoxide, and quantitatively analyzing the content of indigo and indirubin by a method described in the above general experimental method 1 in terms of crude extracts of indigo, refined extracts of crude extracts of indigo, indirubin extracts of indigo and indirubin by high performance liquid chromatography. The whole operation process adopts a brown bottle to avoid light.
Results: as can be seen from the data in table 3, ethyl acetate has good extraction effects on indigo and indirubin, and methanol and ethanol have poor extraction effects on indigo and good extraction effects on indirubin. The extraction effect of methylene dichloride on indigo is poor and the extraction effect of indirubin is general. The invention provides a method for extracting indigotin and indirubin in crude extract fine powder of indigotin in a grading way. Extracting indigo and indirubin from the refined powder of the crude extract of the bluish with ethyl acetate. To obtain ethyl acetate extract. Further extracting the ethyl acetate extract by using an alcohol-soluble solvent to obtain a purer indigo and indirubin extract.
TABLE 3 influence of different solvents on the content of indigo and indirubin in crude extract powder of Kalimeris indica
Example 5: preparation of alkaloid by fractional extraction
Adding 200ml methanol into 20g of crude extract fine powder of herba Kalimeridis, and extracting at 40deg.C for 30min. After centrifugation and secondary extraction with 200ml of methanol, the solvent was recovered in vacuo at low temperature under dark conditions after combining the extracts, and the concentrate was adsorbed on 20ml of methanol 5g silica gel for half an hour. Eluting with 20ml methanol, filtering, eluting for three times, mixing the eluates, and vacuum recovering solvent at low temperature under dark condition to obtain indirubin extract. Adding 200ml of ethyl acetate into the methanol extraction residue in the above steps, and extracting by ultrasonic treatment at 40 ℃ for 30min. Adding 200ml ethyl acetate for secondary extraction by centrifugal filtration, combining the extracting solutions, and vacuum recovering the solvent at a low temperature under dark condition to obtain the indigo extract. Adding 200ml of DMF into the ethyl acetate extraction residue in the above steps, and extracting by ultrasonic treatment at 40 ℃ for 30min. And (3) adding 200ml of DMF (dimethyl formamide) for secondary extraction by centrifugal filtration, combining the extracting solutions, and recycling the solvent in vacuum at a low temperature under dark condition to obtain the tryptanthrin extract. The ranges of the levels of extracted indigo and indirubin are shown in table 4.
TABLE 4 fractional extraction of indigo and indirubin content
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (1)
1. The method for conducting, regulating and controlling the acidification of the crude extract of the bluish with electric conduction and extracting the bluish alkaloid in a grading way is characterized by comprising the following steps:
s1, preparing a crude extract suspension of the bluish red: adding 2-10 times of water into Ma Landian crude extract for dilution, stirring for 0.1-1 hour at the stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to 8000-1.2 ten thousand (mu s/cm), centrifugally filtering, and adding 2-5 times of water into filter residues for dilution to obtain a bluish crude extract suspension;
s2, acidifying a crude extract suspension of the bluish red: adding 10% -25% hydrochloric acid solution into the crude extract suspension of the bluish, acidifying at 40-80 ℃, stirring for 0.5-2 hours at a stirring speed of 100-1000r/min, regulating the electric conductivity of filtrate to be 6-10 ten thousand (mu s/cm), adding 2-5 times of hot water into filter residues, stirring and cleaning for 1-5 times, regulating the electric conductivity of the filtrate to be 1000-5000 (mu s/cm), filtering to obtain acidified filter residues, drying, grinding into powder with an average particle size of 20-180 mu m, and obtaining refined powder of the bluish crude extract;
s3, preparing indirubin extract: adding an alcohol-soluble solvent into the refined powder of the crude extract of the bluish in the step S2 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying filter residues, merging extract liquid, then carrying out low-temperature vacuum recovery of the solvent at the dark, adsorbing concentrate by 5-15 times of polar medium, adding 10-20 times of polar solvent into the concentrate, washing for 1-5 times, filtering, merging washing liquid, and carrying out low-temperature vacuum recovery of the solvent to obtain the indirubin extract with the indirubin content of 10% -45%;
s4, preparing an indigo extract: adding a medium-polarity solvent into the filter residue dried in the step S3 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times under the condition of avoiding light, carrying out centrifugal filtration, drying the filter residue, merging the extract solutions, carrying out low-temperature vacuum recovery of the solvent under the condition of avoiding light, and drying the concentrate into powder to obtain an indigo extract with the indigo content of 20% -60%;
s5, preparing a bluish amine ketone extract: adding a polar solvent into the filter residue in the step S4 according to the solid-to-liquid ratio of 1:3-1:10, carrying out ultrasonic reflux extraction at the extraction temperature of 30-70 ℃ for 1-5 times, carrying out centrifugal filtration, drying the filter residue, merging the extract liquid, carrying out vacuum recovery of the solvent under the dark condition, and drying the concentrate into powder to obtain the bluish amine ketone extract with the tryptanthrin content of more than 15%;
the crude extract of the indigotin is indigotin dry powder or indigo powder prepared by the traditional fermentation process of the stem and leaf of the indigotin, wherein the content of the indigotin is 0.5-7%, the indigotin is 0.1-0.5%, and the tryptanthrin is 0.1-0.2%;
the alcohol-soluble solvent is one of methanol, ethanol and propanol, the medium-polarity solvent is one of ethyl acetate, chloroform and dichloromethane, and the polar solvent is one of DMF and DMSO;
the ultrasonic wave adopts 50hz, and the proportion of ultrasonic power and crude extract of the bluish is 100-800W: 5-50 g;
the light shielding is to isolate a solvent system of the bluish active substance from sunlight, LED light and UV light by using dark vessels or cloths such as black, brown and the like;
the low-temperature vacuum is at 50-60 ℃ and the vacuum degree is 500-760 mmHg, and the polar medium is silica gel.
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| CN102174009A (en) * | 2011-03-09 | 2011-09-07 | 中国科学院近代物理研究所 | Method for preparing indigo and indirubin from dyers woad leaf |
| CN109364126A (en) * | 2018-10-17 | 2019-02-22 | 华侨大学 | A kind of preparation method of indigo naturalis |
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| JP2005255662A (en) * | 2004-05-11 | 2005-09-22 | Kokuhi Tei | Plant component mixture for beverage extraction, mixed liquor of plant component and plant component mixture |
| CN102174009A (en) * | 2011-03-09 | 2011-09-07 | 中国科学院近代物理研究所 | Method for preparing indigo and indirubin from dyers woad leaf |
| CN109364126A (en) * | 2018-10-17 | 2019-02-22 | 华侨大学 | A kind of preparation method of indigo naturalis |
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