阅读说明：本技术 一种以马蓝鲜叶为原料制备青黛的方法 (Method for preparing indigo naturalis from fresh leaves of kalanchoe ) 是由 赵亮 刘兆宇 于 2019-11-20 设计创作，主要内容包括：一种以马蓝鲜叶为原料制备青黛的方法,其特征在于,包括以下步骤：(1)浸泡液预处理；(2)浸泡发酵；(3)打靛-靛合成；(4)粗靛精制；(5)制备成品。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。(A method for preparing indigo naturalis by taking fresh leaves of India tinctoria as a raw material is characterized by comprising the following steps: (1) pretreating a soaking solution; (2) soaking and fermenting; (3) performing beat indigo-indigo synthesis; (4) refining crude indigo; (5) and preparing a finished product. 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. A method for preparing indigo naturalis by taking fresh leaves of India tinctoria as a raw material is characterized by comprising the following steps: (1) pretreating a soaking solution; (2) soaking and fermenting; (3) performing beat indigo-indigo synthesis; (4) refining crude indigo; (5) and preparing a finished product.

2. A method for preparing indigo naturalis from the fresh leaves of India tinctoria as a raw material, which is characterized by comprising the following steps:

(1) pretreatment of a soak solution: mixing deionized water, a leaf stalk of the kalimeris indica and old branches of the kalimeris indica according to a weight ratio of 10-15: 1-3: 1-3, crushing the mixture to 100-200 meshes, and fermenting the mixture in a fermentation tank for 2-3 days;

(2) soaking and fermenting: weighing fresh kale leaves with the weight ratio of 10-15% of the soaking solution in the step (1), washing, cutting into pieces of 1-2cm, adding the pieces of 1-2cm into the soaking solution fermented in the step (1), adding alkali to adjust the pH value to 5.5, soaking for 12h at 30 ℃, stirring for one time for 2h, and after the soaking fermentation is finished, sieving the soaking solution by a sieve to remove impurities;

(3) indigo-indigo synthesis: adding NaOH into the filtrate obtained in the step (2) to adjust the pH value of the filtrate, introducing oxygen, adding a precipitator, stirring, beating indigo for 40min, standing until precipitation, and re-filtering and recovering the upper layer liquid;

(4) refining crude indigo: adding 2 times of water into the precipitate, grinding and washing by using a stirrer, filtering by using a filter membrane to remove impurities, and centrifugally dewatering by using a centrifugal machine to obtain indigo paste;

(5) preparing a finished product: drying the indigo paste at 60 ℃, crushing and sieving by a 100-mesh sieve to obtain the indigo naturalis.

3. The method for preparing indigo naturalis from fresh leaves of India Maka as raw material according to claim 2, wherein in step (3), the pH of the filtrate is adjusted to 10.5, oxygen is introduced for 1h, and the precipitator is lime milk added with 4% of fresh water.

Technical Field

The invention belongs to the field of natural product extraction, and particularly relates to a method for preparing indigo naturalis by taking fresh leaves of India Makino as a raw material.

Background

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. The traditional processing method comprises the following steps: harvesting stems and leaves in autumn, placing the stems and leaves in a soaking pool, introducing river water, completely submerging the stems and leaves, soaking and fermenting for several days, fishing out residues when the pool liquid is dirty green, adding lime, fully stirring, standing for 1-2 days, discarding supernatant, collecting sediment at the bottom, transferring the sediment to a small pool, standing, and discarding supernatant to obtain a semi-finished product, namely crude indigo. And (5) refining the crude indigo through impurity removal and refining processes such as water refining, foam flotation and the like, and drying to obtain the indigo naturalis. [ Jia thinking 21232, chickeny, WUWU [ M ]. Miao inspired to make good results, Miao Longdao, Beijing: agricultural press, 1982: 53.] [ Song should Star. Nature's opener [ M ]. Beijing: commercial bookmarking house, 1954: patent application No. ] patent application No.: 200310110926.8, title of the invention: the patent application of 'a modern production method for processing indigo naturalis' discloses a modern production method for processing crude indigo by using leaves or stems and leaves of a natural plant, namely, India tinctoria Makino: in the soaking step, the cut stems and leaves of the kalimeris indica are added with pretreated soaking water according to the proportion of 1: 10-1: 30, the mixture is soaked and fermented until the soaking solution becomes dirty green, the temperature of the soaking water is 20-50 ℃, the soaking time is 24-72 hours, and then the stem and leaf fragments are filtered; adding quicklime into the crude indigo preparation link according to the proportion that the ratio of the usage amount of the quicklime to the amount of the indigo is 10: 1, blending into lime milk, introducing the lime milk into the soaked fermentation liquor with impurities removed, fully stirring by using a stirrer, and simultaneously increasing or decreasing the usage amount of the quicklime through testing the pH value of the solution in the stirring process until the indigo is observed to have linear precipitation after 1 hour; or directly introducing air into the soaking fermentation tank by using an air blower or an air compressor until the indigo is generated as linear precipitate. On one hand, the pH value test refers to observing the color change of the solution in the pool during stirring, and if the color is changed from green to egg yolk, the lime consumption is proper; on the other hand, for the sake of stability, it is also necessary to verify the taste by mouth, and it is moderately alkaline, i.e., pH 12 to 13, to feel bitter first and then sweet.

The soaking hydrolysis link is started from microbial fermentation, microbes propagate in large quantities under the conditions of proper temperature, value and the like, saccharifying enzymes secreted by fermentation lead glycoside bonds of indole glycosides to be subjected to enzymolysis and breakage, and the microbial metabolic process in the soaking process of indigo naturalis belongs to mixed bacteria fermentation, namely, a method for fermenting by using microbes existing under natural conditions does not artificially add microbial strains, and can produce glucosidase only after a long time. The microorganism producing beta glucosidase is thermophilic bacteria beta, trichoderma, aspergillus niger and the like. Aspergillus niger is a kind of cellulase, and can accelerate the decomposition of the cell wall of the leaf of kale and dissolve out the precursor substance in the plant cell. The lignification degree of the blue old branches and the petioles is larger than that of leaf cells, and the microbial enzyme decomposed cells are dissolved in the indigo substance and take longer time. Further pretreatment can increase yield.

The soaking process is an important part in the whole indigo naturalis production process, and directly influences the dissolution effect of the precursor substance and the conversion of the precursor substance to the indigo and the indirubin. Relating to the yield and quality of the product. The prior indigo preparation process takes a great deal of time in the process, but often cannot fully utilize the raw materials, thereby causing a great deal of waste. The water consumption and the soaking time of soaking have important influence on the quality and the yield of the crude indigo. Indigo naturalis mainly contains effective components indigo blue and indirubin which are converted from precursor substances and need to be acted by enzymes generated by microorganisms. The indigo leaf stalks and old branches contain less indigo precursors, so that the microbial proliferation in the indigo leaves can shorten the soaking time of the fresh indigo leaves, and researches show that the soaking time is prolonged, and the content of indigo and indirubin in indigo is reduced. Therefore, the soaking liquid is treated in advance, the production period of the indigo can be shortened, and the yield of the indigo can be improved.

In addition, the soaking time, temperature, pH and indigo beating process conditions in the preparation process of indigo naturalis all influence the generation of indigo blue and indirubin, and the processing process is difficult to standardize, so that the quality of the finished indigo naturalis is uneven, and the clinical curative effect is influenced. The standardized and modern indigo naturalis processing process quantification is established by combining the traditional processing method, the formulating work of the indigo naturalis standard is promoted, namely, the stable-quality indigo naturalis is obtained through the standardized management of the whole process processing of medicinal material production, and the scientificity and rationality of formulating the indigo naturalis standard are ensured.

Disclosure of Invention

Based on the defects in the background art, the invention intends to disclose a method for preparing indigo naturalis by taking the fresh leaves of India Makino as a raw material, and the extraction efficiency of the indigo naturalis is increased.

Through pretreatment of a petiole and a mallow old branch soaking solution, the main effective components of indigo naturalis, namely indigo and indirubin, are converted from precursor substances by virtue of the action of enzymes generated by microorganisms. The indigo leaf stalks and old branches contain less indigo precursors, so that the microbial proliferation in the indigo leaves can shorten the soaking time of the fresh indigo leaves, the soaking time is prolonged, and the content of indigo and indirubin in the indigo is reduced, so that the production period of the indigo can be shortened and the yield of the indigo can be improved by treating the soaking solution in advance.

The soaking hydrolysis link is started from microbial fermentation, microbes propagate in large quantities under the conditions of proper temperature, value and the like, saccharifying enzymes secreted by fermentation lead glycoside bonds of indole glycosides to be subjected to enzymolysis and breakage, and the microbial metabolic process in the soaking process of indigo naturalis belongs to mixed bacteria fermentation, namely, a method for fermenting by using microbes existing under natural conditions does not artificially add microbial strains, and can produce glucosidase only after a long time. The microorganism producing beta glucosidase is thermophilic bacteria beta, trichoderma, aspergillus niger and the like. Aspergillus niger is a cellulase which can accelerate the decomposition of the cell wall of the leaf of kale and dissolve the substance in the plant cell.

The lignification degree of the blue old branches and the petioles is larger than that of leaf cells, and the microbial enzyme decomposed cells are dissolved in the indigo substance and take longer time. Further pretreatment can increase yield.

The oxygen content in the solution is increased by aeration and stirring, the indigo precursor is promoted to be converted into an intermediate for generating the indigo and the indirubin, and the content of the indigo and the indirubin is increased. In the traditional process, lime milk is added to make the soaking solution alkaline, the lime milk has another function of generating calcium carbonate which is an indigo indirubin adsorption carrier in sedimentation, and the use amount of calcium carbonate can be reduced by adjusting the pH value with NaOH, so that the later-stage impurity removal and purification of indigo naturalis are facilitated.

The invention is realized by the following process steps:

a method for preparing indigo naturalis from fresh leaves of India tinctoria comprises the following steps: (1) pretreating a soaking solution; (2) soaking and fermenting; (3) performing beat indigo-indigo synthesis; (4) refining crude indigo; (5) and preparing a finished product.

Preferably, the process comprises the following specific steps:

(1) pretreatment of a soak solution: mixing deionized water, a leaf stalk of the kalimeris indica and old branches of the kalimeris indica according to a weight ratio of 10-15: 1-3: 1-3, crushing the mixture to 100-200 meshes, and fermenting the mixture in a fermentation tank for 2-3 days;

(2) soaking and fermenting: weighing fresh kale leaves with the weight ratio of 10-15% of the soaking solution in the step (1), washing, cutting into pieces of 1-2cm, adding the pieces of 1-2cm into the soaking solution fermented in the step (1), adding alkali to adjust the pH value to 5.5, soaking for 12h at 30 ℃, stirring for one time for 2h, and after the soaking fermentation is finished, sieving the soaking solution by a sieve to remove impurities;

(3) indigo-indigo synthesis: adding NaOH into the filtrate obtained in the step (2) to adjust the pH value of the filtrate, introducing oxygen, adding a precipitator, stirring, beating indigo for 40min, standing until precipitation, and re-filtering and recovering the upper layer liquid;

(4) refining crude indigo: adding 2 times of water into the precipitate, grinding and washing by using a stirrer, filtering by using a filter membrane to remove impurities, and centrifugally dewatering by using a centrifugal machine to obtain indigo paste;

(5) preparing a finished product: drying the indigo paste at 60 ℃, crushing and sieving by a 100-mesh sieve to obtain the indigo naturalis.

Preferably, in the step (3), the pH of the filtrate is adjusted to 10.5, oxygen is introduced for 1h, and the precipitator is lime milk added with 4% of fresh water.

The invention has the advantages of

(1) By pretreating the petiole and the branch of Indigofera tinctoria soaking solution, the microorganism proliferation in the leaves of Indigofera tinctoria can shorten the soaking time of the fresh leaves of Indigofera tinctoria, and the pretreatment of the soaking solution can shorten the production period of indigo naturalis and improve the conversion rate of the indigo naturalis.

(2) The oxygen content in the solution is increased by aeration and stirring, the indigo precursor is promoted to be converted into an intermediate for generating indigo and indirubin, the content of the indigo and indirubin is increased, the use amount of calcium carbonate can be reduced by adjusting the PH by using NaOH, the later-stage impurity removal and purification of the indigo are facilitated, and the indigo yield is improved.

Detailed Description

Example 1

Influence of different soaking treatment and fermentation time on indigo naturalis preparation

In the fermentation process of the fresh leaves of the kalimeris indica, the soaking condition and the soaking time of the raw materials are key factors influencing the quality and the yield of the indigo naturalis. At a proper temperature, the soaking time is too short, the fermentation is insufficient, the soaking time is too long, the main effective component indigo blue indirubin of the indigo naturalis is degraded, and the indigo precursor is converted into other indigo substances. The fermentation time can be shortened by properly increasing the temperature, and the reproduction of microorganisms and the activity of enzymes can be influenced by overhigh temperature, so that the indigo and indirubin generated by the indigo precursor substance can be influenced. According to the fermentation principle and data analysis test, for scientific and standardized production, the fermentation of the fresh leaves of the kalimeris indica is carried out at 30 ℃ under stirring and semi-oxygen permeation.

The influence of the pretreatment of the soak solution on the yield and the quality of the indigo naturalis is in the embodiment. Weighing 10 parts of fresh leaves of kalanchoe indica collected in September, each part is 1kg, cleaning, and cutting into 1.5 CM. 5 groups are soaked in 30 degrees of 30 water for 12, 24, 36, 48 and 72 hours respectively.

The other five groups are soaked in 30 times of 30-degree soaking solution for 3 days for 4, 8, 12, 24 and 36 hours. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust pH of the filtrate to 10.5, introducing air for 1 hr, adding 4% fresh lime milk, stirring, beating indigo for 40min, standing for precipitation, filtering the supernatant, recovering, and mixing the precipitates. And detecting the yield of the product indigo indirubin by an RP-HPLC method.

TABLE 1 Effect of different fermentation times on the yield of indigo and indirubin

Fermentation time (h)	Indigo (g/kg)	Indirubin (g/kg))
			Malan blue leaf fresh product	0.48	4.15
12	3.45	3.86
			24	8.84	4.35
36	12.57	5.04
			48	15.82	5.58
72	14.73	5.12

TABLE 2 influence of different fermentation times after the pre-fermentation treatment of the soaking solution on the yield of indigo and indirubin

The weight of the raw medicinal materials of each group is calculated by fresh products. From the data in tables 1 and 2, it was found that the yield of indigo and indirubin was 16.32g/kg and 5.58g/kg, respectively, using conventional kalium marianum fresh leaf soaking fermentation at 30 ℃ for an optimal time of 48 hours. The method is characterized in that the red indigo is soaked and fermented in the fresh leaves of the red indigo after the pre-soaking liquid is adopted for fermentation treatment, the optimal soaking time is 12 hours under the temperature condition of 30 ℃, the yield of the red indigo is 17.28g/kg and the yield of the red indigo is 6.27g/kg, the red indigo is saved by 36 hours compared with the conventional fermentation, and the content of the red indigo can be improved. Therefore, the fresh leaves and stems of the kalimeris indica and old branches are adopted for fermentation in advance, the production period is shortened without increasing the cost, and the yield and the production efficiency are improved.

Example 2

Content of indigo-indirubin in ramulus et folium Lespedezae Cuneatae

Weighing 3 parts of 1kg of blue old branches collected in September, cleaning, and cutting into 1-2 CM. 3 groups are soaked in 30 degrees of 30 water for 48 hours, 72 hours and 84 hours respectively. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust pH of the filtrate to 10.5, introducing air for 1 hr, adding 4% fresh lime milk, stirring, beating indigo for 40min, standing for precipitation, filtering the supernatant, recovering, and mixing the precipitates. And detecting the yield of the product indigo indirubin by an RP-HPLC method.

TABLE 3 detection of product indigo-indirubin yield

Example 3

Influence of oxygen on indigo naturalis preparation (increasing indigo indirubin yield)

1kg of the kale was soaked for 12 hours at 30 degrees with the soak solution fermented with the old petioles in advance using the optimal soaking fermentation mode in example 1. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust the pH value of the filtrate to 10.5, introducing air for 1h, adding 4% of newly prepared lime milk, stirring and beating indigo for 40min, standing for precipitation, filtering and recovering supernatant, crushing the precipitate, adding 2 times of water, stirring and washing, centrifugally filtering to remove impurities, drying filter residue at 60 ℃, crushing and sieving with a 100-mesh sieve to obtain 181.33g of finished indigo naturalis, wherein the content of indigo and indirubin in the indigo naturalis is respectively 9.37% and 2.89%.

1kg of the kale was soaked for 12 hours at 30 degrees with the soak solution fermented with the old petioles in advance using the optimal soaking fermentation mode in example 1. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust the pH value of the filtrate to 10.5, adding 4% of newly prepared lime milk without ventilation, stirring and beating indigo for 40min, standing for precipitation, filtering and recovering supernatant, grinding the precipitate, adding 2 times of water, stirring and washing, centrifugally filtering to remove impurities, drying filter residue at 60 ℃, crushing and sieving with a 100-mesh sieve to obtain 148.75g of finished indigo naturalis, wherein the content of indigo blue and indirubin in the indigo naturalis is respectively 6.54% and 1.72%.

Example 4

Preparation of indigo naturalis (higher calcium carbonate content) by adjusting pH with alkali

By adopting the optimal soaking fermentation mode in the embodiment, 1kg of kale is soaked for 12 hours by 30 degrees in the soaking solution fermented by the petioles and the old branches in advance. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust the pH value of the filtrate to 10.5, introducing air for 1h, adding 4% of newly prepared lime milk, stirring and beating indigo for 40min, standing for precipitation, filtering and recovering supernatant, crushing the precipitate, adding 2 times of water, stirring and washing, centrifugally filtering to remove impurities, drying filter residue at 60 ℃, crushing and sieving with a 100-mesh sieve to obtain 181.33g of finished indigo naturalis, wherein the content of indigo and indirubin in the indigo naturalis is respectively 9.37% and 2.89%.

1kg of the kale was soaked for 12 hours at 30 degrees with the soak solution fermented with the old petioles in advance using the optimal soaking fermentation mode in example 1. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Introducing air for 1h, adding 12% of newly prepared lime milk, stirring and beating indigo for 40min, standing for precipitation, filtering and recovering supernatant, crushing the precipitate, adding 2 times of water, stirring and washing, centrifuging, filtering and removing impurities, drying filter residue at 60 ℃, crushing and sieving by a 100-mesh sieve to obtain finished products of 353.28g of indigo naturalis, wherein the content of indigo blue and indirubin in the indigo naturalis is respectively 4.15% and 1.22%.

Example 5

By adopting the optimal soaking fermentation mode in the example 1, 1kg of the kalimeris indica is soaked in the soaking solution fermented by the petioles and the old branches in advance for 12 hours at 30 ℃. Stirring once for 1 hour, filtering the soak solution by a screen to remove impurities. Adding NaOH to adjust the pH value of the filtrate to 10.5, introducing air for 1h, adding 4% of newly prepared lime milk, stirring and beating the indigo for 40min, standing and precipitating, filtering and recovering supernatant, crushing the precipitate, adding 2 times of water, stirring and washing, centrifugally filtering to remove impurities, drying filter residue at 60 ℃, crushing and sieving with a 100-mesh sieve to obtain 181.33g of finished product indigo naturalis, and detecting the content of the indigo and the content of the indirubin in the indigo naturalis to obtain the content of the indigo and the content of the indirubin respectively of 9.37% and 2.89%.