Method for regulating indigo / isatin blue ratio of extract of strobilanthes by acid-base synergistic gas explosion

Abstract

This invention provides a method for regulating the indigo / indirubin ratio of *Indigofera tinctoria* extract using acid-base synergistic aeration. The method involves grading fresh *Indigofera tinctoria* raw materials, removing impurities such as soil at pH 4-7 to obtain clean fresh leaves, followed by hot air stabilization treatment and cold air slow release to obtain dried leaves with high indole glycoside content, achieving stable storage of the fresh *Indigofera tinctoria* leaf alkaloids. Using ultrasonic extraction with an acidic phosphate buffer solution, and employing enzyme-synergistic acidification and alkalization aeration to regulate pH, low-ratio and high-ratio indigofera extracts are obtained, respectively. Finally, a high-ratio indirubin extract is obtained through low-temperature eutectic extraction. The produced high-ratio indigo extract has an indigo:indirubin ratio of 70:1 to 30:1 and an indigo content of 40% to 70%. Compared with traditional indigo paste, the relative ratio of indigo is increased by 6 to 12 times, and its dyeing effect reaches that of synthetic high-ratio indigo products. The high-ratio indirubin extract has an indirubin:indigo ratio of 5:1 to 30:1 and an indirubin content of 10% to 35%. It not only provides a raw material source for the preparation of high-content indirubin, but also has been applied to the dyeing of cotton fabrics for the first time, pioneering the purple-red dyeing system.

Description

Technical Field

[0001] This invention belongs to the field of development of natural plant extracts and their natural antibacterial dyes, and specifically relates to a method for regulating the indigo / indirubin ratio of strychnine extract. Background Technology

[0002] Indigofera tinctoria is a perennial herb native to southeastern and southwestern China, India, Japan, and the Indochina Peninsula. It is both a traditional Chinese medicine used to prepare indigo for thousands of years and a primary raw material for the blue dye indigo, holding a unique place in human history.

[0003] In recent years, the traditional lime-processed indigo and its preparations have shown significant efficacy in treating leukemia, psoriasis, ulcerative colitis, mumps, and other infectious and inflammatory diseases. Preparations primarily composed of indigo include compound indigo tablets, Qingbing powder, indigo powder, compound indigo pills, and compound indigo capsules, among many other tablet, powder, pill, and capsule formulations, with wide applications. The processing of indigo has a long history. Although modern scholars have optimized various parameters in the traditional processing, such as the pH of the soaking solution, the ratio of solvent volume to fresh leaf mass, soaking time, soaking temperature, indigo-removing pH, and the amount of lime milk added, and have used technologies like foam flotation to mechanize production instead of traditional family workshops, the content of indigo and other key components like indigo red and indigo in the resulting indigo is still relatively low. The introduction of lime in the traditional indigo processing, along with the complexity and uncontrollability of the process, leads to inconsistent quality of indigo, affecting its efficacy. This invention aims to develop a chemically directed conversion process based on the processing principle of indigo, to regulate and optimize the formation direction of indigo and indirubin according to market demand, to remove the lime component in indigo, to optimize the quality of indigo products, and to obtain indigo products with high content of effective components such as indigo and indirubin.

[0004] Besides indigo, indigo dye has been used for over 5,000 years. It was even found on the clothing of ancient Egyptian mummies dating back 5,000 years. Records of indigo dyeing techniques from indigo plants in China first appeared in the Northern Wei Dynasty's *Qimin Yaoshu*. Indigo plants were soaked, fermented, and processed with quicklime to produce indigo paste. It was later discovered that adding rice wine or wine lees could ferment and reduce the indigo dye solution, thus ushering in the era of artificial fermentation and reduction dyeing. Indigo plants were transported back to Europe as a valuable "spice," and their excellent color and lightfastness made indigo dye a prized commodity. Until the late 19th century, indigo dye was still derived from indigo plants such as *Indigofera tinctoria*, *Isatis tinctoria*, and *Polygonum tinctorium*, and indigo became a major trade commodity for the Dutch and British East India Companies. In the history of natural dyes, indigo dye was widely used because of the fresh, elegant, bright, and rich colors of the fabrics it produced.

[0005] Since German chemist Adolf von Baeyer discovered the chemical structure of indigo in 1897, and BASF launched the first commercially available synthetic indigo in the same year, synthetic indigo has rapidly replaced plant-based indigo in the dyeing and printing industry due to its lower price, higher purity (over 95%), absence of indirubin, and high indigo ratio (100%). In contrast, traditional lime-based indigo paste contains less than 3% indigo, has a yield of less than 0.2%, and is composed of over 95% inorganic impurities such as calcium oxide, calcium hydroxide, and calcium carbonate. This results in low indigo purity and low colorfastness, and the indigo paste powder contains only 0.1-0.3% indirubin, with an indigo to indirubin ratio of only 5:1-12:1, making it a low indigo ratio product. To improve the indigo content and the indigo to indoruby ratio in natural indigo, the international practice is to soak indigo plants followed by aeration oxidation. This process can achieve an indigo purity of 20-30%, but aeration reduces the stability of indophenol, resulting in an indigo yield of less than 0.1%. In particular, the indigo to indoruby ratio is only 10:1 to 20:1, still considered a low-ratio indigo product. Therefore, regardless of whether lime-based or aeration-based indigo production is used, the low indigo content and relative ratio, coupled with the fact that indoruby affects the brightness of pure blue in dyeing and causes significant red interference, prevent the large-scale application of low-ratio indigo products in modern dyeing and printing industries. These products are only suitable for use in intangible cultural heritage handmade creative clothing. Summary of the Invention

[0006] In view of this, in order to improve the high content and high ratio of natural indigo, solve the problem of high brightness and high color fastness of pure blue in natural plant dyeing, and expand the demand for natural indigo extract in the pharmaceutical, cosmetic, and food industries, this invention innovatively proposes a method for acid-base synergistic aeration to regulate the indigo / indirubin ratio of *Indigofera tinctoria* extract. Through raw material grading, uniform spraying with pH 4-7 acidic water, and hot air treatment, key technologies for the stable storage of alkaloids from fresh *Indigofera tinctoria* leaves are achieved, preparing high-content indole-glycoside *Indigofera tinctoria* raw materials. Then, through extraction, enzyme-synergistic acidification or alkalization reactions, pH adjustment, aeration oxidation, and solvent extraction, extracts with high indigo and indirubin ratios are prepared. The technical solution of this invention includes the following steps: The first step is to grade the fresh raw materials of Malan: harvest fresh raw materials of Malan stems and leaves of 10cm~80cm on the same day, cut off 20cm~50cm of woody stems with a hydraulic cutter, retain 10cm~30cm of tender branches and leaves, and spray them evenly with water with pH 4~7 to remove dirt and other impurities, so as to obtain clean fresh Malan leaves. The second step is the hot air stabilization treatment of fresh leaves of *Indigofera tinctoria*: the fresh leaves of *Indigofera tinctoria* are first blown with hot air at 60℃~80℃ for 5min~20min, then slowly released with cold air at 10℃~20℃ for 20min~30min, then blown with hot air at 250℃~350℃ for 3min~6min, and finally slowly released with cold air at 10℃~20℃ for 20min~30min to obtain dried leaves of *Indigofera tinctoria*, which are then crushed into 5~20 mesh.

[0007] The third step is the extraction of indole glycosides from the leaves of *Indigofera tinctoria*: Fresh or dried leaves of *Indigofera tinctoria* are extracted with a phosphate buffer solution of pH 5-7, with a solid-liquid ratio (g / mL) of 1:5-80. The extraction is performed by ultrasound or microwave for 10-90 minutes at a temperature of 20-60℃. The extraction is repeated twice and then filtered through a 40-100 mesh filter cloth to obtain the extract of *Indigofera tinctoria* leaves. Step 4, Enzyme-Coordinated Acidification and Aeration Reaction: First, add 0.01%~5% (v / v) of a bio-complex enzyme (Aspergillus niger enzyme: glucoamylase mass ratio of 2:1) to the *Ipomoea strychnifolia* leaf extract. Adjust the pH to 5~6 with hydrochloric acid, maintain the temperature at 30℃~60℃, and react for 2~8 hours. Then, adjust the pH to 2~4 with hydrochloric acid again, maintain the temperature at 60℃~80℃, and perform intermittent aeration reaction for 0.5~2 hours. Cool the reaction solution with 0℃~10℃ low-temperature water and let it stand for 4 hours. -10h, filter to obtain indigo extract A; add 0.1%~0.5% NaOH solution to indigo extract A, with a solid-liquid ratio (g / mL) of 1:30~60, adjust the pH of the solution to 10~12, stir at 60℃~80℃ for 10~30min, filter, wash the precipitate with water 2~3 times, centrifuge to obtain indigo extract B, dry to obtain indigo high ratio extract, wherein indigo:indorubicin = 70:1~30:1, and the indigo content is 40%~70%; Step 5, aeration-synergistic alkalization reaction: Adjust the pH of the intermittent aeration reaction solution or filtered liquid from Step 4 to 8-10 using 5% sodium hydroxide solution (pH 2-4). Add 0.01%-0.5% cysteine ​​by mass of the reaction solution volume. Maintain the temperature at 60℃-80℃ and the intermittent aeration reaction time at 0.5-2 hours. Incubate at this temperature for 4-10 hours, centrifuge, and dry the precipitate to obtain a low-ratio indigo extract, wherein the ratio of indigo to indoruby is 3:1-30:1, and the indigo content is 20%-35%. Step 6, Preparation of high-ratio indigo extract: Take the low-ratio indigo extract from step 5, add organic solvent and extract 1-5 times, with a solid-liquid ratio (g / mL) of 1:15-50, and extract with ultrasound or microwave for 10-90 minutes at a temperature of 20℃-50℃. Combine the extracts, centrifuge, concentrate the filtrate under vacuum, and evaporate the concentrate to dryness to obtain the high-ratio indigo extract, wherein the ratio of indigo to indigo is 5:1-30:1, and the indigo content is 10%-35%.

[0008] Mature *Indigofera tinctoria* plants typically grow to a height of 80-120 cm in the Yunnan-Guizhou region, with woody stems comprising two-thirds of the plant. The indigo precursor content is 5-10 times lower than that of fresh leaves, and the harvested material contains a small amount of mud and sand. Therefore, this patent further addresses this issue by employing a stem and leaf grading process to increase the indigo precursor content of fresh raw materials. In laboratory settings, small quantities of raw materials are cut with scissors, while in factory production, a hydraulic cutter is preferred for its speed and large processing capacity. The woody stems are kept to 20-50 cm, and the tender branches and leaves are retained to 10-30 cm. During the grading process, tissue damage leads to the encounter and degradation of glucosidase and indole glycosides. Since indole glycosides are stable in a weakly acidic environment, based on the pH adjustment described in steps one and three, one of citric acid, acetic acid, oxalic acid, sodium dihydrogen phosphate, and hydrochloric acid is used to adjust the pH of the aqueous solution to 5-7, with citric acid, oxalic acid, and acetic acid being preferred for adjusting the pH to 5-6. The fresh, tender branches and leaves graded using this patent method have a water content of 60% to 80% and an indole glycoside content of 1.5% to 2%, while the raw material of *Indigofera tinctoria* that has not undergone grading and pH acid washing has an indole glycoside content of only 0.5% to 1.0%, which is far lower than that of the raw material treated by this method.

[0009] Furthermore, in step two above, the stabilization treatment of fresh *Indigofera tinctoria* leaves at 250℃~350℃ is preferably achieved by drying at 280℃~300℃ for 3 min~6 min, followed by slow release with 10℃ cold air for 30 min, resulting in dried *Indigofera tinctoria* leaves with a moisture content controlled below 8%, crushed to 5~20 mesh, and containing 8%~12% indole glycosides. In contrast, the indole glycoside content of raw materials dried directly in the sun or shade is only 0.2%~0.5%, that of raw materials dried at 50℃~100℃ is only 0.5%~3.4%, that of raw materials dried at 100℃~200℃ is 2.5%~5.5%, and that of raw materials dried at 300℃ is 8.0%~10%, which is significantly higher than other drying methods.

[0010] Furthermore, the indole glycoside extraction from senna leaves described in this invention, whether using fresh or dried senna leaves, preferably employs ultrasonic or microwave extraction with a phosphate buffer solution at pH 5-7. The solid-liquid ratio (g / mL) for fresh senna leaves is 1:5-40, and for dried senna leaves it is 1:40-80. Ultrasonic extraction is preferably performed for 10-30 minutes at a temperature of 20-30°C, and the extraction is repeated twice. The extract is then filtered through a 40-60 mesh filter to obtain the senna leaf extract.

[0011] Furthermore, in the enzyme-synergistic acidification aeration reaction described in this invention, the preferred bio-complex enzyme is Aspergillus niger enzyme:saccharifying enzyme with a mass ratio of 2:1, the enzyme concentration is preferably 0.5%~1%, the preferred temperature is 50℃~60℃, the pH is adjusted to 5~6 with hydrochloric acid, and under the controlled weakly acidic pH conditions, the bio-complex enzyme acts for a reaction time preferably 8 hours to rapidly degrade indoleglycosides into indophenol. To overcome the problem of incomplete degradation, the pH is further adjusted to 3 with hydrochloric acid, and the reaction temperature is 80℃ to promote the complete degradation of indoleglycosides in the extract into indophenol. Then, air or ozone is preferably aerated at the bottom of the reaction solution for 2-5 minutes, with intermittent aeration every 10 minutes, for a reaction time of 0.5 hours, to promote the oxidative polymerization of indophenol to generate indigo and indirubin. Further addition of 0.5% NaOH solution, with a preferred solid-liquid ratio (g / mL) of 1:30~40, and adjustment of the solution pH to 10~12, followed by stirring at 60℃ for 30 min, can remove a large amount of alkali-soluble impurities from the precipitate. After washing with water 2~3 times, a high-ratio indigo extract is obtained, in which the ratio of indigo to indorubicin is 70:1~30:1, and the indigo content is 40%~70%, which can meet the quality requirements of natural indigo in the standardized modern printing and dyeing industry.

[0012] Furthermore, to efficiently convert indole glycoside extract into high-quality indigo, the effects of ozone time, acid hydrolysis time, hydrochloric acid concentration, and pH on the precipitation yield and the content of indigo and indirubin were investigated. A four-factor, three-level experiment was designed using SPSS software to optimize the directional indigo production process. Specifically, the aeration or ozone time was 3 min, the hydrochloric acid concentration was 2%, the acid hydrolysis time was 1.0 h, and the pH was 7. Under these process conditions, the indigo content and total indigo yield could reach 46–60% and 2–4%, respectively.

[0013] Furthermore, the aeration-assisted alkalization reaction described in this invention obtains a low-ratio indigo extract under alkaline pH conditions. First, the pH of the reaction solution or filtered liquid from the fourth step (intermittent aeration, pH 2-4) is adjusted to pH 8-10. Cysteine ​​is then added, followed by intermittent aeration with air or ozone for 2-5 minutes, with an aeration oxidation cycle every 10 minutes for a total reaction time of 0.5 hours. The purpose is to stabilize indophenol to prevent degradation and simultaneously promote the formation of indirubin, increasing its proportion. After standing at an alkaline pH for 8-10 hours, a low-ratio indigo extract is obtained, wherein the indigo:indirubin ratio is 3:1-30:1, and the indigo content is 20%-35%. Eutectic solvents, also known as eutectic mixtures or deep eutectic solvents, are a novel type of ionic liquid analogue. They possess unique physicochemical properties and numerous advantages, including low cost, simple preparation process, non-toxicity, no vapor pressure, and easy biodegradability. Furthermore, by adjusting the structure and ratio of their components, they can be endowed with specific functionalities and tunability, making them a more environmentally friendly type of solvent. Deuterated eutectic solvents (DES) are eutectic mixtures with melting points below 100°C, formed by hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) through intermolecular hydrogen bonds. HBAs are primarily quaternary ammonium salts and quaternary phosphate salts, such as choline chloride and methyltrioctylammonium chloride, while HBDs are mainly amides, alcohols, and carboxylic acids, such as n-propanol, palmitic acid, myristic acid, lauric acid, and ricinoleic acid.

[0014] Furthermore, the preparation of the high-ratio indigo extract of the present invention uses the low-ratio indigo extract from step five as raw material, and adds any one of the following organic solvents: methanol, anhydrous ethanol, ethyl acetate, acetone, chloroform, and a eutectic solvent. Methanol, acetone, and a eutectic solvent are preferred. The extraction is performed ultrasonically 2-3 times, with a solid-liquid ratio (g / mL) of 1:25-30, for 10-30 minutes. The eutectic solvent is further specified as methyltrioctylammonium chloride:palmitic acid:octanoic acid in a molar ratio of 100:60:40. The mixture is reacted ultrasonically or microwaved for 30-60 minutes at a temperature of 20-30°C to obtain the high-ratio indigo extract, wherein the indigo:indigo ratio is 5:1-30:1, and the indigo content is 10%-35%.

[0015] To improve the dyeing effect of indigo extracts with different ratios, this patent selects indigo extracts with a high ratio, wherein the ratio of indigo to indorubicin is 70:1 to 50:1, the indigo content is 60% to 70%, the dyeing strength (for standard products) is 100 points, the K / S value is greater than 20, and the dyeing performance and fastness on cotton fabrics are: dyeing depth grade 1, color fastness to soaping grade 4 to 5, color fastness to perspiration grade 4 to 5, color fastness to dry rubbing grade 4 to 5, and color fastness to water grade 4 to 5. The preferred high-ratio extract of indigo red, wherein the ratio of indigo red to indigo is 20:1 to 30:1, the indigo red content is 20% to 35%, the purple-red dyeing strength (of the standard) is 100 points, the K / S value is greater than 20, and the dyeing performance and fastness on cotton fabrics are: dyeing depth grade 1, color fastness to washing with soap grade 4 to 5, color fastness to perspiration grade 4 to 5, color fastness to dry rubbing grade 4 to 5, and color fastness to water grade 4 to 5.

[0016] The present invention has the following advantages: pH was controlled by enzyme-synergistic acidification and aeration, and aeration-synergistic alkalization to obtain low-ratio indigo extract, high-ratio indigo extract, and high-ratio indirubin extract, respectively.

[0017] Traditional lime-based or aeration-based indigo products have an indigo to indirubin ratio of only 5:1 to 12:1, resulting in low indigo ratio products. This invention, through raw material grading, pH adjustment, and aeration processes, produces a high-ratio indigo extract with an indigo:indirubin ratio of 70:1 to 30:1 and an indigo content of 40% to 70%. This represents a 6-12 fold increase in the relative indigo ratio, achieving dyeing effects comparable to high-ratio synthetic indigo products. In the printing and dyeing industry, it provides a bright, free-flowing blue color with good colorfastness, meeting the quality requirements of natural indigo in modern, standardized printing and dyeing industries.

[0018] From the low-ratio extract of indigo, a high-ratio extract of indigo red was obtained through low-temperature eutectic extraction, wherein the ratio of indigo red to indigo is 5:1 to 30:1 and the content of indigo red is 10% to 35%. This not only provides a raw material source for the preparation of high-content indigo red, but also, for the first time, it is applied to the dyeing of cotton fabrics, thus pioneering the purple-red dyeing system. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Stems and leaves of *Isatis indigotica*, graded and cleaned fresh leaves, and dried leaves Figure 2 Appearance of Indigo Extract from Fresh Senna Leaves at Different Ratios Figure 3 HPLC of Indigo Extract from Fresh Senna Leaves at Different Ratios Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Grading Experiment of Fresh Strobilanthes bidentata Raw Materials Take 20 kg of fresh *Indigofera tinctoria* stems and leaves harvested on the same day, with a length of 10 cm to 80 cm. Remove 20 cm to 50 cm of woody stems, retaining 10 cm to 30 cm of tender branches and leaves. The moisture content should be 60% to 80%, and the indole glycoside content 1.5% to 2%. Use one of the following: citric acid, acetic acid, oxalic acid, sodium dihydrogen phosphate, or hydrochloric acid, preferably citric acid and sodium dihydrogen phosphate, to adjust the pH of the aqueous solution to 4 to 7. Spray evenly to remove dirt and other impurities, yielding 7 kg of cleaned fresh *Indigofera tinctoria* leaves. Fresh leaves of *Indigofera tinctoria* are first dried by blowing with hot air at 60℃~80℃ for 5min~20min, followed by slow release with cold air at 10℃~20℃ for 20min~30min, then by blowing with hot air at 250℃~350℃ for 3min~6min, preferably 300℃, until the moisture content is below 15%. Finally, they are dried with cold air at 10℃~20℃ for 20min~30min to obtain 1kg of dried leaves of *Indigofera tinctoria*. These leaves are then crushed into 5~20 mesh pieces, with a moisture content below 8% and indole glycosides of 8%~12%. In comparison, the indole glycoside content of raw materials dried directly in the sun or shade is only 0.2%~0.5%, that dried at 50℃~100℃ is only 0.5%~3.4%, that dried at 100℃~200℃ is 2.5%~5.5%, and that dried at 300℃ is 8.0%~10%.

[0023] Table 1 Comparison of indole glycoside content in fresh raw materials of *Indigofera tinctoria* after grading and processing tender branches and leaves of Malan 75%~85% 0.35%~1.0% 0.04%~0.08% tender branches of Malan 5%~12% 0.05%~0.08% 0.01%~0.05% Fresh leaves of Malan 75%~85% 1.2%~2.5% 0.05%~0.1% Dried leaves of indigo 5%~8% 8%~12% 0.5%~1.0% Microwave-dried senna leaves 5%~8% 10%~12% 0.1%~0.3% Dried leaves of Malan 8%~12% 0.2%~0.5% 0.4%~0.8% Dried leaves of Malan 10%~15% 0.2%~0.5% 0.2%~0.5% Dried leaves of *Indigofera tinctoria* at 50℃ 8%~12% 0.5%~3.4% 0.5%~1.0% Dried leaves of *Indigofera tinctoria* at 300℃ 6%~10% 8%~12% 0.5%~1.0% Example 2: Preparation of high-ratio extract of Indigofera tinctoria Take 1 kg of fresh leaves of *Indigofera tinctoria* or 0.3 kg of dried leaves, and extract with a phosphate buffer solution with a pH of 5-7. The solid-liquid ratio (g / mL) is 1:5-80, preferably 1:15-30 for fresh leaves and 1:60-80 for dried leaves. Extract with ultrasound or microwave for 10-90 min, preferably 30 min, at 30℃, twice. Filter with a 60-mesh filter cloth to obtain 20-30 L of *Indigofera tinctoria* leaf extract. Add 0.3%–0.5% (v / v) of a bio-complex enzyme (Aspergillus niger enzyme: glucoamylase in a 2:1 mass ratio) to the extract of *Ipomoea strychnifolia* leaves. Adjust the pH of the extract to 5–6 with hydrochloric acid, and maintain the temperature at 30–50°C, preferably 40°C, for 4 hours. Then, adjust the pH to 2–3 with hydrochloric acid, maintain the temperature at 60°C, and perform intermittent aeration with air or ozone for approximately 2–5 minutes, interrupting the aeration every 10 minutes, for a total of 0.5 hours. Cool the reaction solution to 10°C with 0°C water. After standing for 6 hours, the mixture was filtered to obtain indigo extract A and 25 L of clarified fermented compound bacterial solution. Indigo extract A was added to 0.2% NaOH solution at a solid-liquid ratio of 1:35 (g / mL), and the pH was adjusted to 10-12. The solution was stirred at 60℃ for 30 minutes, filtered, and the precipitate was washed 2-3 times with water and centrifuged to obtain indigo extract B. After drying, 6-8 g of high-ratio indigo extract was obtained, in which the ratio of indigo to indorubicin in the fresh leaves of *Indigofera tinctoria* was 40:1-30:1, and the indigo content was 40%-50%. The ratio of indigo to indorubicin in the dried leaves of *Indigofera tinctoria* was 70:1-60:1, and the indigo content was 50%-70%. The product was a black powder, as shown in the image. Figure 2 The test results of five batches of samples of Indigofera tinctoria extract from fresh leaves are shown in Table 2.

[0024] Table 2. Detection of Indigo Extract from Fresh Leaves of Malanjing (n=5) 0.72 5.68 4.35 67.62 1.86 36.35 Example 3: Preparation of low-ratio extract of Indigofera tinctoria Take 20L of the aerated clarified liquid from the compound bacterial fermentation in Example 2, adjust the pH to 8-10 (preferably pH 9) with 5% sodium hydroxide solution, add 0.01%-0.5% cysteine ​​(preferably 0.1%-0.3%) by volume of the clarified liquid, adjust the temperature of the clarified liquid to 60℃-80℃, and then aerate intermittently with air or ozone for about 2-5 minutes, interrupted every 10 minutes, for a reaction time of 1 hour. Keep warm and let stand for 6 hours, centrifuge to obtain precipitate and 20L of pH 9 aerated clarified liquid. Dry the precipitate to obtain 12-13g of indigo low-ratio extract, wherein the indigo:indorubicin ratio is 6:1-15:1, the indigo content is 20%-35%, and the appearance is a black powder. The test results of 5 batches of samples of the indigo low-ratio extract from fresh leaves of *Indigofera tinctoria* are shown in Table 3.

[0025] Table 3. Detection of low-ratio extracts of indigo from fresh leaves of *Indigofera tinctoria* (n=5) 1.24 4.89 2.82 26.46 2.37 11.16 Example 4: Preparation of high-ratio indigo extract DES is generated by reacting different hydrogen bond acceptors and different hydrogen bond donors in a specific molar ratio. HBA is mainly composed of quaternary ammonium salts and quaternary phosphate salts, such as choline chloride and methyltrioctylammonium chloride, while HBD is mainly composed of amides, alcohols, and carboxylic acids, such as n-propanol, palmitic acid, myristic acid, lauric acid, and ricinoleic acid. In this example, the reaction temperature was selected as 80 °C, and the reaction stirring time was 5 h. Observing the properties of DES, the optimal ratio of methyltrioctylammonium chloride:palmitic acid:n-octanoic acid was selected as 2.5:1.5:1, resulting in a golden-yellow liquid with excellent fluidity. See Table 4. Table 4. Molar ratios and property molar ratios of DES with different compositions DES-8 Methyltrioctylammonium chloride: Lauric acid 1:2 pale yellow solid DES-9 Methyltrioctylammonium chloride: n-Decanonic acid 1:2 Pale yellow liquid with good fluidity DES-10 Methyltrioctylammonium chloride: Ricinoleic acid 1:2 Brownish-red liquid with good fluidity DES-11 Choline chloride: urea 5:7 Crystals precipitate out; white solid DES-12 Choline chloride: citric acid 1:1 Colorless liquid with extremely poor fluidity DES-13 Choline chloride: sucrose 2:1 Layered structure, poor liquidity DES-14 Choline chloride: Ricinol acid 1:1 Layered structure, poor liquidity DES-15 Choline chloride: hexanoic acid 1:1 White solid DES-16 Choline chloride: n-propanol 1:2 Crystals precipitate, white solid DES-17 Methyltrioctylammonium chloride: n-Caulic acid: Ricinoleic acid 1:1:1 Golden yellow liquid with good fluidity DES-18 Methyltrioctylammonium chloride: Palmitic acid: Octyl acid 2.5：1.5：1 Golden yellow liquid with excellent fluidity Then, using the low-ratio indigo extract from the examples as raw material, approximately 10g was weighed for each experiment and added to organic solvents such as methanol, anhydrous ethanol, ethyl acetate, acetone, and chloroform, respectively, at a solid-liquid ratio (g / mL) of 300 ml. Ultrasonic extraction was performed for 30 min. The indigo and indirubin contents were determined by HPLC. The indirubin content ranged from 10% to 30%, with an indirubin:indigo ratio of 3:1 to 8:1. Chloroform extraction showed the best results, yielding an indirubin content of 27.79% and indigo content of 3.42%, with an indirubin:indigo ratio of 8:1, as shown in Table 5. Table 5. Effects of different organic solvents on the extraction of indigo red. dichloromethane 3.86 21.24 6.77 12.79 Ethyl acetate 2.28 13.40 7.23 12.98 acetone 2.41 12.47 8.83 12.33 Anhydrous ethanol 3.41 10.97 8.86 13.47 chloroform 3.42 27.79 7.06 12.55 In this embodiment, a eutectic solvent of methyltrioctylammonium chloride:palmitic acid:octanoic acid = 100:60:40 was selected, and the temperature was 25℃. Centrifugation was performed, and the supernatant was collected. The lower solid layer was further extracted by ultrasonication for 30 min. 600 ml of the two supernatants were combined. The organic solvent was concentrated, and the concentrate was evaporated to dryness to obtain 2-3 g of indigo red high-ratio extract. The indigo and indigo red content was determined by HPLC. The indigo red content was 35%, with an indigo red:indigo ratio of 10:1. The product appeared as a pink powder. The results of the determination of five batches of indigo red high-ratio extract from fresh leaves of *Indigofera tinctoria* are shown in Table 6.

[0026] Table 6. Detection of high ratio of indigo to red extract from fresh leaves of *Indigofera tinctoria* (n=5) 0.26 3.55 1.56 3.46 35.77 10.33 Example 5: Evaluation of staining with indigo extracts at different ratios Samples of high-ratio indigo extract (67.2% indigo, 36.35% indigo to indirubin), high-ratio indirubin extract (35.77% indirubin, 10.33% indigo to indigo), and synthetic indigo (99.8% indigo content) were weighed separately and thoroughly shaken and dissolved in a room temperature shake dyeing tester. DM-1228 penetrant and sodium bisulfite were added and stirred thoroughly. The pH was adjusted to 10.5 with sodium hydroxide solution to obtain the dyeing solution. The fabric (40s*40s / 133*72 pure cotton poplin) was first treated with 10 g / L DM-1228 penetrant (two dips and two nips), then padded in the dyeing solution. The dyed fabric was exposed to air and gently shaken by hand to ensure full oxidation. Finally, it was squeezed with water (three dips and three nips), removed, dried, and compared using a computer colorimeter to obtain the K / S value. The washing fastness of the test samples was tested according to ISO 105-C10:2006. The rubbing fastness was analyzed using an electrical rubbing fastness tester according to ISO 105-X12:2016.

[0027] The K / S value is the ratio of the pigment absorption coefficient k to the pigment scattering coefficient s, and is usually used to indicate the depth of color on the surface of a solid sample, i.e., the dyeing rate. A higher K / S value means better dyeing performance. Calculations show that a high-ratio indigo extract, with a dyeing strength (of the standard) of 100 points and a K / S value greater than 20, exhibits dyeing performance and fastness on cotton fabrics of dyeing depth grade 1, washing fastness to soaping grade 4-5, perspiration fastness grade 4-5, dry rubbing fastness grade 4-5, and water fastness grade 4-5. This high-ratio indigo extract achieves the dyeing effect of synthetic indigo. Similarly, a high-ratio indigo extract, with a purple-red dyeing strength (of the standard) of 100 points and a K / S value greater than 20, exhibits dyeing performance and fastness on cotton fabrics of dyeing depth grade 1, washing fastness to soaping grade 4-5, perspiration fastness grade 4-5, dry rubbing fastness grade 4-5, and water fastness grade 4-5. Therefore, its application in dyeing cotton fabrics has expanded the purple-red dyeing system.

[0028] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The solutions disclosed in the embodiments are described simply because they correspond to the methods disclosed in the embodiments; relevant parts can be found in the method section.

[0029] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for acid-base synergistic aeration regulation of the indigo / indirubin ratio in strychnine extract, characterized in that... Includes the following steps: The first step is grading the fresh raw materials of *Indigofera tinctoria*: Fresh raw materials of *Indigofera tinctoria* stems and leaves, 10cm-80cm long, are harvested on the same day. The woody stems of 20cm-50cm are removed using a hydraulic cutter, leaving 10cm-30cm of tender branches and leaves. The leaves are then evenly sprayed with water of pH 4-7 to remove dirt and other impurities, resulting in clean fresh *Indigofera tinctoria* leaves. The second step is hot air stabilization treatment of the clean fresh *Indigofera tinctoria* leaves: The clean fresh *Indigofera tinctoria* leaves are first blown with hot air at 60℃-80℃ for 5min-20min, then slowly released with cold air at 10℃-20℃ for 20min-30min, then blown with hot air at 250℃-350℃ for 3min-6min, and finally slowly released with cold air at 10℃-20℃ for 20min-30min, resulting in dried *Indigofera tinctoria* leaves, which are then crushed into 5-20 mesh. Step 3: Extraction of indole glycosides from *Indigofera tinctoria* leaves: Fresh or dried *Indigofera tinctoria* leaves are extracted with a phosphate buffer solution at pH 5-7, with a solid-liquid ratio (g / mL) of 1:5-80. The extraction is performed ultrasonically or microwaved for 10-90 minutes at a temperature of 20-60℃, and repeated twice. The extract is then filtered through a 40-100 mesh filter to obtain the *Indigofera tinctoria* leaf extract. Step 4: Enzyme-synergistic acidification and aeration reaction: 0.01%-5% (v / v) of a bio-complex enzyme (Aspergillus niger enzyme: saccharifying enzyme mass ratio of 2:1) is added to the *Indigofera tinctoria* leaf extract. The pH is adjusted to 5-6 with hydrochloric acid, and the temperature is maintained at 30-60℃ for 2-8 hours. The pH was adjusted to 2-4 with hydrochloric acid, the temperature was maintained at 60℃-80℃, and the intermittent aeration reaction time was 0.5-2 hours. The reaction solution was cooled with water at 0℃-10℃ and allowed to stand for 4-10 hours. After filtration, indigo paste A was obtained. Indigo paste A was added to 0.1%-0.5% NaOH solution at a solid-liquid ratio (g / mL) of 1:30-60. The pH of the solution was adjusted to 10-12, and the mixture was stirred at 60℃-80℃ for 10-30 minutes. After filtration, the precipitate was washed with water 2-3 times and centrifuged to obtain indigo paste B. After drying, a high-ratio indigo extract was obtained, in which the ratio of indigo to indorubicin was 70:1-30:1 and the indigo content was 40%-70%. Step 5, aeration-assisted alkalization reaction: Adjust the pH of the intermittent aeration reaction solution or filtered liquid from Step 4 to 8-10 using 5% sodium hydroxide solution (pH 2-4). Add 0.01%-0.5% cysteine ​​by mass of the reaction solution volume. Maintain the temperature at 60℃-80℃ and the intermittent aeration reaction time at 0.5-2 hours. Incubate at this temperature for 4-10 hours, centrifuge, and dry the precipitate to obtain a low-ratio indigo extract with an indigo:indorubicin ratio of 3:1-30:1 and an indigo content of 20%-35%. Step 6, Preparation of high-ratio indigo extract: Take the low-ratio indigo extract from step 5, add organic solvent and extract 1-5 times, with a solid-liquid ratio (g / mL) of 1:15-50, and extract with ultrasound or microwave for 10-90 minutes at a temperature of 20℃-50℃. Combine the extracts, centrifuge, concentrate the filtrate under vacuum, and evaporate the concentrate to dryness to obtain the high-ratio indigo extract, wherein the ratio of indigo to indigo is 5:1-30:1, and the indigo content is 10%-35%.

2. The method for acid-base synergistic aeration regulation of the indigo / indirubin ratio in strychnine extract according to claim 1, characterized in that... The pH value mentioned in steps one and three is adjusted to a water pH of 5-7 using one of the following: citric acid, acetic acid, oxalic acid, or sodium dihydrogen phosphate.

3. The method for acid-base synergistic aeration regulation of the indigo / indirubin ratio in strychnine extract according to claim 1, characterized in that... The first-stage grading of fresh tender branches and leaves has a water content of 60%~80% and an indole glycoside content of 1.5%~2%.

4. The method for acid-base synergistic aeration regulation of the indigo / indirubin ratio in strychnine extract according to claim 1, characterized in that... The dried leaf powder of *Isatis tinctoria* obtained in the second step has a moisture content of 5% to 10% and an indole glycoside content of 6% to 12%.

5. The method for acid-base synergistic aeration regulation of the indigo / indirubin ratio in strychnine extract according to claim 1, characterized in that... The organic solvent for the sixth step is any one of methanol, anhydrous ethanol, ethyl acetate, acetone, chloroform, or a eutectic solvent.

6. The indigo high-ratio extract according to claim 1, characterized in that... Natural indigo is a deep blue color with a dyeing strength (for standard products) of 100 points and a K / S value greater than 20. Its dyeing performance and fastness on cotton fabrics are: dyeing depth grade 1, color fastness to soaping grade 4-5, color fastness to perspiration grade 4-5, color fastness to dry rubbing grade 4-5, and color fastness to water grade 4-5.

7. The high-ratio extract of indigo according to claim 1, characterized in that... Natural indigo red is purple or purplish-red. Its purplish-red dyeing strength (for standard products) is 100 points, and its K / S value is greater than 20. Its dyeing performance and fastness on cotton fabrics are: dyeing depth grade 1, color fastness to washing with soap grade 4-5, color fastness to perspiration grade 4-5, color fastness to dry rubbing grade 4-5, and color fastness to water grade 4-5.

8. The eutectic solvent according to claim 5, characterized in that... The eutectic solvent is composed of methyltrioctylammonium chloride, palmitic acid, and octanoic acid in a molar ratio of 100:60:

40. The reaction is carried out using ultrasound or microwave for 10 to 90 minutes at a temperature of 20°C to 50°C.

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