阅读说明：本技术 马蓝鲜叶连续制备青黛、靛蓝、靛玉红的方法 (Method for continuously preparing indigo naturalis, indigo and indirubin from fresh leaves of kale ) 是由 赵亮 刘兆宇 于 2019-11-20 设计创作，主要内容包括：马蓝鲜叶连续制备青黛、靛蓝、靛玉红的方法,其特征在于,由以下步骤组成：(1)青黛制备；(2)青黛中提取靛蓝、靛玉红；(3)靛蓝、靛玉红粗品除杂；(4)靛蓝、靛玉红分离纯化；(5)靛蓝衍生物食用靛蓝制备。通过对叶柄、马蓝老枝浸泡液预处理,提前使马蓝叶内微生物增殖能缩短马蓝鲜叶浸泡时间,提前处理浸泡液能缩短青黛生产周期,及提高青黛转化率,通气搅拌使溶液中氧气含量增加,促进靛前体物质转化为生成靛蓝、靛玉红的中间体,增加靛蓝靛玉红的含量,利用NaOH调节PH可以减少碳酸钙的用量,便于青黛后期除杂纯化,提高青黛得率。(The method for continuously preparing indigo naturalis, indigo and indirubin from fresh leaves of kale is characterized by comprising the following steps: (1) preparing indigo naturalis; (2) extracting indigo and indirubin from indigo naturalis; (3) removing impurities from crude indigo and indirubin products; (4) separating and purifying the indigo and the indirubin; (5) indigo derivatives are prepared from edible indigo. Through pretreatment of a petiole and a Chinese cabbage old branch soaking solution, microbial proliferation in a Chinese cabbage leaf can be shortened through the pretreatment, the Chinese cabbage fresh leaf soaking time can be shortened through the pretreatment of the petiole and the Chinese cabbage old branch soaking solution, the natural indigo production period can be shortened through the pretreatment of the Chinese cabbage old branch soaking solution, the natural indigo conversion rate can be improved through the pretreatment of the Chinese cabbage fresh leaf soaking solution, the oxygen content in the solution is increased through aeration and stirring, the indigo precursor substance is promoted to be converted into an intermediate for generating the natural indigo and the indirubin, the content of the indigotin indirubin is increased, the use amount of calcium carbonate can be reduced through the regulation of PH.)

1. The method for continuously preparing indigo naturalis, indigo and indirubin from fresh leaves of kale is characterized by comprising the following steps: (1) preparing indigo naturalis; (2) extracting indigo and indirubin from indigo naturalis; (3) removing impurities from crude indigo and indirubin products; (4) separating and purifying the indigo and the indirubin; (5) indigo derivatives are prepared from edible indigo.

2. A method for continuously preparing indigo naturalis, indigo blue and indirubin by taking fresh leaves of Indigofera tinctoria as a raw material according to claim 1, which is characterized by comprising the following specific steps:

(1) preparing indigo naturalis: weighing fresh kale leaves, cutting into small sections with the length of 1cm, washing, placing in a clean vessel, adding 20 times of clear water, soaking for 48 hours at 30 ℃, after soaking and fermentation, enabling a soak solution to pass through a 10-mesh screen firstly, then filtering and removing impurities through a 40-mesh screen, weighing lime with the weight of 6% of the raw materials, adding a proper amount of water, uniformly stirring to form lime milk, adding the lime milk into the soak solution, placing in a stirrer, stirring for 40 minutes, refrigerating, standing and precipitating for 24 hours, pouring out a filtrate to obtain crude indigo, adding water into the crude indigo to prepare crude indigo pulp with the concentration of 2.5%, filtering by a filter membrane, centrifugally dewatering at 1000r/min, drying for 10 hours at 60 ℃ until the water content is less than 7.0%, and crushing and sieving by a 100-mesh sieve to obtain the kal;

(2) extracting indigo and indirubin from indigo naturalis: adding 4 times of 95% ethanol by mass equivalent into a natural indigo finished product, stirring, performing ultrasonic treatment for 30min, performing suction filtration, extracting for three times, combining three filtrates, performing rotary evaporation, concentration and recovery of an ethanol solvent, adding distilled water with the same amount as the ethanol after concentrating to 1/3, performing cooling filtration after concentrating and recovering 1/2 water, and obtaining filter residues, namely crude indigo and indirubin;

(3) removing impurities from crude indigo and indirubin: taking the dried filter residue for extracting the indigo and the indirubin, adding 6 times of 95% ethanol by weight equivalent for extraction, pouring out a solution after the extraction is finished, carrying out reduced pressure concentration, recovering the ethanol to obtain a reddish brown paste, adding 3 times of water, adding hydrochloric acid to make the solution acidic, keeping the pH value at 3-4, refrigerating, standing and precipitating for 24 hours, filtering, adjusting the solution back to alkaline pH 7-9, passing the filtrate through a macroporous resin column, washing with 6 times of water to be nearly colorless, then eluting with ethyl acetate and ethanol, and carrying out reduced pressure concentration on the eluate;

(4) separating and purifying the indigo and the indirubin by a chromatographic column: mixing crude indigo, indirubin and alkaline alumina with the same amount of powder, stirring, loading the powder 10 times the weight equivalent of alumina into column, loading sample, covering 1CM thick alumina on the upper layer, and filling a layer of absorbent cotton on the upper layer. Firstly, mixing ethyl acetate: and (3) spreading by using 80% ethanol in a ratio of 3:1, separating two color bands of blue and purple, collecting the blue color band which is the indigo when the first drop of the spreading agent flows out, and using 100% ethanol after the blue color band is collected: eluting with ethyl acetate at 3:2 flow rate of 1.5d/s, and collecting a purple red color band. Concentrating the collected blue eluate and mauve eluate under reduced pressure, recrystallizing with methyl ethanol, filtering under reduced pressure, recovering ethanol, washing with a small amount of water to remove impurities, and drying to obtain blue crystal, i.e. indigo blue and mauve crystal, i.e. indirubin;

(5) preparation of indigo derivative edible indigo: weighing indigo and indirubin, drying and crushing filter residues obtained by suction filtration during extraction, adding the fine powder into concentrated sulfuric acid with 2-3 times of fine powder, heating and stirring for 1h, cooling, adding water with 10 times of concentrated sulfuric acid for dilution, adding sodium bicarbonate to adjust the pH value to 4, adding 12% sodium chloride for salting out, slowly stirring to completely dissolve, standing for 30min, filtering, washing the filter residues with water, and drying to obtain the edible indigo.

3. The method for continuously preparing indigo naturalis, indigo naturalis and indirubin from fresh leaves of kale in claim 2, wherein the pH of the soaking solution in the step (1) is 3.5, and the fermentation process is micro-aerobic fermentation, wherein the oxygen content is 20%.

4. The method for continuously preparing indigo naturalis, indigo naturalis and indirubin from fresh leaves of kale according to claim 2, wherein the indigo naturalis and indigo naturalis are prepared by adjusting the pH of soaking solution to 10.5 with strong alkali sodium hydroxide or potassium hydroxide, heating the solution to 60 ℃, and stirring for 40min by ventilation.

5. The method for continuously preparing indigo naturalis, indigo blue and indirubin from fresh leaves of Indigofera tinctoria of claim 2, wherein the step (2) of extracting the Indigofera tinctoria from the finished product of indigo naturalis adopts an ultrasonic-assisted extraction method, 95% ethanol with 4 times of indigo naturalis volume is added into the indigo naturalis, the mixture is uniformly stirred, the mixture is subjected to ultrasonic treatment at 250W for 30min, the filtration is carried out, the filter residue is re-extracted by 95% ethanol, and the ultrasonic-assisted extraction is carried out for three times.

Technical Field

The invention belongs to the field of natural product extraction, and particularly relates to a method for preparing indigo by taking fresh leaves of kalanchoe odorata 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. Radix Isatidis, stem and leaf can be used as raw materials, and its stem and leaf are processed into a dark blue powder as a Chinese medicinal material, named as indigo naturalis, with cold nature and salty taste, and also has effects of clearing away heat and toxic materials, cooling blood, and arresting convulsion. Indigo (Indigo) and Indirubin (Indirubin) are effective components of Indigo naturalis, wherein Indigo is widely used for dyes and food pigments and has a certain protection effect on liver. Indirubin is a novel double-indole anti-tumor drug which is discovered from natural indigo of Chinese herbal medicine and has a treatment effect on chronic granulocytic leukemia (lentil), and is an anti-leukemia drug with a novel structure which is pioneered in China.

Indigo naturalis, indigo naturalis and indirubin are prepared from "blue" plants such as Indigowoad blue, isatis tinctoria and Polygonum tinctoria, wherein the indirubin has the highest content in fresh Indigowoad blue leaves, meanwhile, the Indigowoad blue stem leaves contain a large amount of indirubin or free indoxyl serving as an indirubin precursor substance, and the finished Indigowoad blue processed by the Indigowoad stem leaves has high content of the indirubin and better quality than other similar products.

Natural indigo, name of Chinese traditional medicine. Is prepared from leaf or stem of Acanthaceae plant herba Strychni, Polygonum tinctorium L of Polygonaceae, and Isatis tinctoria of Cruciferae by processing to obtain dry powder, mass or granule. Has the functions of clearing away heat and toxic material, cooling blood and removing ecchymoses, purging fire and arresting convulsion, and is also commonly used for printing and dyeing cloth, eyebrow penciling and the like in ancient times. Mainly produced in Fujian, Yunnan and Jiangsu provinces. The nature and taste are salty and cold, and the medicine has the efficacies of clearing away heat and toxic material, cooling blood and removing ecchymoses, purging fire and arresting convulsion and the like. It is mainly used for treating epidemic febrile disease with excessive heat, macula, hematemesis, hemoptysis, pharyngalgia, aphtha, infantile convulsion, sore, swelling, erysipelas, snake and insect bite, etc. Indigo is prepared by processing indigo blue, a large amount of rich processing and application experiences are accumulated in the application history of thousands of years, indigo is gradually developed into a medicine from a dye, and the indigo blue is also developed into an indigo-making raw material from an indigo-making raw material. The traditional processing of indigo naturalis mainly comprises three links of soaking, indigo beating, refining and the like, and the processing process mainly converts precursor substances such as indole glycoside, isatin B and the like in fresh leaves or stems and leaves of blue plants into effective components such as indigo, indirubin and the like.

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.

Indigo is the main component of Chinese medicine indigo naturalis, and has effects of resisting inflammation and protecting liver. Indigo-based pigments are one of the oldest pigments known to humans, and have been extracted from plants of the bluegrass family, are water-soluble non-azo colorants, are used as edible pigments in the form of their sodium sulfonate salts or their aluminized forms, and are widely used in the food, pharmaceutical, and printing and dyeing industries. Indigo was originally extracted from plants and later developed to be prepared synthetically by chemical synthesis. The plant extraction method comprises the following steps: and (3) piling the indigo leaves, and watering frequently to ferment the indigo leaves for 2-3 months to form black soil blocks. The ball indigo is called ball indigo after being tamped by a mortar, and contains 2 to 10 percent of indigo pigment. Mixing wood ash, lime and bran into the indigo globulifera, adding water, mixing, heating to 30-40 ℃, and exposing in air to obtain blue insoluble indigo, wherein the yield is low. The method for preparing the indigo by chemical synthesis pollutes the environment and has potential carcinogenic risk, so the method for preparing the indigo by plants has higher development prospect.

Disclosure of Invention

Based on the defects in the background art, the invention aims to disclose a method for preparing indigo naturalis, indigo and indirubin by using fresh leaves of kale as raw materials, so that the comprehensive utilization rate of the kale is increased, and the production benefit is improved.

Malan blue is a shrub-like perennial herb of Acanthaceae, and its root, stem and leaf can be used as medicine. The fresh leaves of the indigo naturalis can be used for preparing indigo naturalis, the main effective components of the indigo naturalis are indigo and indirubin, but the indigo and the indirubin are not completely and directly existed in a plant body of the indigo naturalis, only a small part of the indigo is directly existed in the plant body, and the stem leaves contain a large amount of indoside or free indoxyl which is a precursor substance of the indigo and the indirubin. The pH, temperature, soaking liquid volume, oxygen amount and the like of the fresh leaves of the kalanchoe indicus are controlled, the directional conversion of the indigo precursor substance into the indigo and the indirubin is promoted, and the yield of the indigo and the indirubin in the indigo is improved.

After high-yield and high-quality indigo naturalis is prepared, the indigo indirubin is extracted from the indigo naturalis by ethanol through an ultrasonic-assisted extraction method, an ethanol extraction agent is cheap, safe, environment-friendly and easy to recover, and the ultrasonic-assisted extraction method is quick, simple and efficient to operate. The indigo naturalis waste residue which is not fully extracted is sulfonated for preparing edible indigo, so that the waste is reduced, and the comprehensive utilization rate is improved.

The method is realized by the following process steps:

the method for continuously preparing indigo naturalis, indigo and indirubin from fresh leaves of kale comprises the following steps: (1) preparing indigo naturalis; (2) extracting indigo and indirubin from indigo naturalis; (3) removing impurities from crude indigo and indirubin products; (4) separating and purifying the indigo and the indirubin; (5) indigo derivatives are prepared from edible indigo. The method for continuously preparing indigo naturalis, indigo and indirubin by taking the fresh leaves of the kalanchoe as raw materials comprises the following specific steps:

(1) preparing indigo naturalis: weighing fresh kale leaves, cutting into small sections with the length of 1cm, washing, placing in a clean vessel, adding 20 times of clear water, soaking for 48 hours at 30 ℃, after soaking and fermentation, enabling a soak solution to pass through a 10-mesh screen firstly, then filtering and removing impurities through a 40-mesh screen, weighing lime with the weight of 6% of the raw materials, adding a proper amount of water, uniformly stirring to form lime milk, adding the lime milk into the soak solution, placing in a magnetic stirrer, stirring for 40 minutes, refrigerating, standing and precipitating for 24 hours, pouring out a filtrate, obtaining crude indigo, adding water into the crude indigo to prepare crude indigo pulp with the concentration of 2.5%, filtering by a filter membrane, centrifugally dewatering at 1000r/min, drying for 10 hours at 60 ℃ until the water content is less than 7.0%, and crushing and sieving by a 100-mesh sieve to obtain the kal;

(2) extracting indigo and indirubin from indigo naturalis: adding 4 times of 95% ethanol by mass equivalent into a natural indigo finished product, stirring, performing ultrasonic treatment for 30min, performing suction filtration, extracting for three times, combining three filtrates, performing rotary evaporation and concentration to recover an ethanol solvent, concentrating to an original volume of 1/3, adding distilled water with the same amount as the ethanol, concentrating to recover 1/2 of water, cooling, and filtering to obtain filter residues, namely crude indigo and indirubin;

(3) removing impurities from crude indigo and indirubin: taking the dried filter residue for extracting the indigo and the indirubin, adding 6 times of 95% ethanol by weight equivalent for extraction, pouring out the solution after the extraction is finished, decompressing and concentrating, recovering the ethanol to obtain a reddish brown paste, adding 3 times of water, adding hydrochloric acid to make the solution acidic, keeping the pH value at 3-4, refrigerating and standing for 24h for precipitation, filtering, adjusting the solution back to alkalescent pH 7-9, passing the filtrate through a macroporous resin column, washing with 6 times of water to be nearly colorless, then eluting with ethyl acetate and ethanol, decompressing and concentrating the eluent.

(4) Separating and purifying the indigo and the indirubin by a chromatographic column: mixing crude indigo, indirubin and alkaline alumina with the same amount of powder, stirring, loading the powder 10 times the weight equivalent of alumina into column, loading sample, covering 1CM thick alumina on the upper layer, and filling a layer of absorbent cotton on the upper layer. . Firstly, mixing ethyl acetate: and (3) spreading by using 80% ethanol in a ratio of 3:1, separating two color bands of blue and purple, collecting the blue color band which is the indigo when the first drop of the spreading agent flows out, and using 100% ethanol after the blue color band is collected: eluting with ethyl acetate at 3:2 flow rate of 1.5d/s, and collecting a purple red color band. Concentrating the collected blue eluate and mauve eluate under reduced pressure, recrystallizing with methyl ethanol, filtering under reduced pressure, recovering ethanol, washing with a small amount of water to remove impurities, and drying to obtain blue crystal, i.e. indigo blue and mauve crystal, i.e. indirubin;

(5) preparation of indigo derivative edible indigo: weighing indigo and indirubin, drying and crushing filter residues obtained by suction filtration during extraction, adding the fine powder into concentrated sulfuric acid with 2-3 times of fine powder, heating and stirring for 1h, cooling, adding water with 10 times of concentrated sulfuric acid for dilution, adding sodium bicarbonate to adjust the pH value to 4, adding 12% sodium chloride for salting out, slowly stirring to completely dissolve, standing for 30min, filtering, washing the filter residues with water, and drying to obtain the edible indigo.

Preferably, in the method for preparing indigo by using the fresh leaves of kalanchoe as the raw material, the pH of the soak solution in the step (1) is 3.5, the fermentation process is micro-aerobic fermentation, and the oxygen content is controlled to be 20%. Preferably, the indigo beating process in the step (1) is to adjust the pH of the soaking solution to 10.5 by using strong alkali sodium hydroxide or potassium hydroxide, heat the solution to 60 ℃, and ventilate and stir for 40 min.

Preferably, the indigo indirubin is extracted from the indigo naturalis finished product in the step (2), an ultrasonic-assisted extraction method is adopted, 95% ethanol with 4 times of indigo naturalis volume is added into the indigo naturalis, the mixture is uniformly stirred, ultrasonic treatment is carried out at 250W for 30min, filtration is carried out, the filter residue is re-extracted by 95% ethanol, and ultrasonic-assisted extraction is carried out for three times.

The invention has the advantages of

(1) The preparation method of indigo naturalis is simple, low cost, high yield of indigo naturalis, and high content of indigo blue and indirubin.

(2) The production of edible indigo turns waste into useful, increases benefit and reduces waste.

(3) The whole process flow is simple, integrated, continuous, high in comprehensive utilization rate and high in preparation yield of the indigo naturalis, the indigo blue and the indirubin. But also sulfonating the indigo generated in the extraction process and not extracted and remained in the waste residue to produce the edible indigo, thereby changing waste into valuable.

Detailed Description

Example 1

Weighing 1kg of fresh kale leaf, cutting into sections of about 1cm, washing, and placing in a clean vessel. Adding 30 times of clear water, soaking at 30 deg.C for 48 hr, stirring once for 2 hr, fermenting for a period of time, passing the soaking solution through a 10 mesh screen, and then filtering through a 40 mesh screen to remove impurities. Weighing 6% lime, adding appropriate amount of water, stirring to obtain lime milk, and adding into the soaking solution. Placing in a stirrer, ventilating and stirring for 40min, refrigerating, standing for precipitation for 24h, and pouring out the filtrate (for detection and recovery) to obtain crude indigo. Adding water into crude indigo to make into crude indigo slurry with concentration of 2.5%, filtering with filter membrane (centrifuging at 1000 r/min), dewatering, drying at 60 deg.C for 10 hr (water content is not more than 7.0%), pulverizing, and sieving with 100 mesh sieve to obtain 225.46g of indigo naturalis, with indigo blue and indirubin content of 7.31% and 1.68%, respectively.

Example 2

Weighing 500g of a certain amount of indigo naturalis finished product prepared by the method in example 1, adding 4 times of 95% ethanol, stirring, performing ultrasonic treatment for 30min, performing suction filtration (keeping filter residue for preparing edible indigo), and extracting for three times. Mixing the three filtrates, rotary evaporating, concentrating, recovering ethanol solvent, and adding distilled water equal to ethanol. Concentrating and recovering 1/2 water, cooling, and filtering to obtain residue (crude product 89.36 g) containing 35.84% and 12.69% of indigo and indirubin respectively.

Separating and purifying by a chromatographic column: mixing crude indigo blue and indirubin with appropriate amount of alkaline alumina, stirring, loading 10 times of alumina into column, covering small amount of alumina on the upper layer after sample application, and adding small amount of absorbent cotton. Firstly, mixing ethyl acetate: and (3) developing by using 80% ethanol in a ratio of 3:1, and separating two color bands of blue and purple. When the first drop of developer comes out, the blue band is collected, i.e. indigo. After collecting the blue color band, 100% ethanol is used: eluting with ethyl acetate at 3:2 flow rate of 1.5d/s, and collecting a purple red color band. Concentrating the collected blue eluate and mauve eluate under reduced pressure, recrystallizing with methanol, filtering under reduced pressure, recovering ethanol, washing with a small amount of water to remove impurities, and drying to obtain blue crystal and mauve crystal. 28g of indigo is obtained, and the purity of the indigo is 97.5%. 8g of indirubin is obtained, and the purity is higher than 98%.

Example 3

1kg of fresh leaves of Indigofera tinctoria is prepared into indigo naturalis by the method in example 1, filter residues obtained by suction filtration when extracting indigo and indirubin from indigo naturalis with ethanol are collected, the filter residues are dried and crushed, fine powder is added into a certain amount of concentrated sulfuric acid, the mixture is heated and stirred for 1 hour, water with 10 times of concentrated sulfuric acid is added for dilution after cooling, sodium bicarbonate is added to adjust the pH value to 4, 12% sodium chloride is added for salting out, standing is carried out for 30min after all the fine powder is slowly stirred and dissolved, filtering is carried out, the filter residues are washed for 3 times with water, and 12g of edible indigo is obtained finally after drying, wherein the purity is 98%.