Integrated preparation method of strobilanthes cusia indigo extract and phenolic acid extract

Abstract

In the process of producing indigo naturalis by taking strobilanthes cusia as a raw material, a very unpleasant smell is generated, a large amount of alkaline wastewater is discharged, and serious environmental pollution is caused. Therefore, development of a more environment-friendly production process and a higher value-added strobilanthes cusia extract is extremely urgent. The invention innovatively provides a green and efficient production process, namely integrally producing the indigo strobilanthes extract and the phenolic acid extract with higher additional values through endogenous enzymatic conversion coupled membrane separation and freeze drying technologies. Through an endogenous enzyme conversion process, the indigo extract with indigo content 20 times and indirubin content 1.8 times higher than that of traditional indigo can be prepared within one day. The phenolic acid extract is recovered from the indigo extract production wastewater by adopting membrane separation and freeze drying processes, and meanwhile, zero discharge of the wastewater can be realized. The prepared indigo extract shows a more excellent dyeing effect on pure cotton poplin than an Indian natural indigo extract. In addition, the prepared phenolic acid extract shows excellent inhibitory activity (IC50 = 32 [mu] g / mL) on staphylococcus aureus, and the wound healing rate of mice exceeds 98%. The method lays a foundation for high-valued and comprehensive utilization of strobilanthes cusia resources, and is of great significance to extension and sustainable and healthy development of a strobilanthes cusia industrial chain.

Description

Technical Field This invention belongs to the field of dye, daily chemical and functional feed additive development, and specifically relates to an integrated preparation method of indigo extract and phenolic acid extract. Background Technology

[0001] Indigofera tinctoria is a perennial herb native to southeastern and southwestern China, India, Japan, and the Indochina Peninsula. As a primary raw material, it has been used to prepare indigo for thousands of years. The traditional production process of indigo is as follows: Indigofera tinctoria is soaked in a pool for 3-7 days for natural fermentation. Then, the indigofera tinctoria residue is removed, lime is added, and the fermentation liquid is stirred. Indigo is obtained through separation, filtration, and drying. Indigo has various medicinal values, including clearing heat and detoxifying, cooling blood and stopping bleeding, and clearing liver heat. Indigo is also an ancient plant dye widely used in printing and dyeing processes. When combined with other natural dyes, it can produce a wider range of color effects. The 2020 Chinese Pharmacopoeia provides specific requirements and explanations regarding the characteristics, identification methods, moisture content, water-soluble pigments, and the content and detection methods of indigo and indirubin indigo. Indigo has become an important source of income for local ethnic minorities and enterprises in many major Indigofera tinctoria growing areas in China. However, the above-mentioned production process of indigo produces a very unpleasant odor and discharges large amounts of alkaline wastewater, leading to the closure of many workshops and enterprises by local governments. Therefore, it is urgent to develop more environmentally friendly production processes and higher value-added indigo extract.

[0002] Indigo extract is another important natural dye, and its preparation process is similar to that of indigo. The indigo content in indigo extract is dozens of times higher than that indigo. Furthermore, as the most important natural blue dye, indigo extract holds a virtually unique position in the field of natural dye textile dyeing. Compared to chemically synthesized dyes, indigo extract produces a more natural and softer color, and has better lightfastness and wash resistance. Therefore, indigo extract is more expensive than both natural and synthetic indigo. The production of indigo extract in India is industrialized, mainly used in the dye industry. However, research on indigo extract in my country is relatively limited, and there are no precedents for industrial-scale production. Zheng et al. developed an efficient process for producing indigo extract from *Indigofera tinctoria* leaves and successfully implemented a pilot-scale verification. In recent years, the team at the Nanjing Institute of Forestry Chemistry, Chinese Academy of Forestry, has conducted some exploratory work on indigo extract. For example, Zhang et al. provided a chemical conversion method to prepare indigo extract from dried *Indigofera tinctoria* leaves, achieving indigo purity and yield of 42.6% and 2.26%, respectively. Chen et al. prepared an indigo extract with an indigo content of 37.4% and an indirubin content of 2.30% using an exogenous enzymatic conversion method, and found that the prepared indigo extract retained the same chemical composition as conventional indigo. Chemical and exogenous enzymatic conversion methods are effective in preparing indigo extracts, but these methods still have drawbacks such as high hydrochloric acid consumption and expensive exogenous enzymes.

[0003] It has been reported that *Indigofera tinctoria* leaves contain abundant active ingredients. Sun et al. summarized compounds isolated from *Indigofera tinctoria* leaves, including alkaloids, flavonoids, organic acids, glycosides, sterols, pentacyclic triterpenes, anthraquinones, amino acids, and sugar compounds. Xue et al. analyzed these compounds using HPLC-IT-TOF-MS. 2 The chemical composition of *Indigofera tinctoria* leaves was analyzed, identifying nine major phenolic compounds, including three phenolic acids (neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid), two phenylethanol glycosides (verbenosinoside and isorhamnetin), and four flavonoids (isorhamnoside-3-O-glucoside, lantanaside, isorhamnoside, and trichomein). Polyphenols, organic acids, polysaccharides, and flavonoids are water-soluble components of *Indigofera tinctoria* leaves. During the preparation of indigo extract, these components dissolve in the extract and remain in the post-fermentation wastewater. In fact, these components possess good potential for antibacterial, wound-healing, antioxidant, and antitumor activity. Unfortunately, in actual production, these components are discharged along with the wastewater. Therefore, finding a green and efficient method to prepare indigo extract while simultaneously recovering these active ingredients is crucial for the comprehensive and high-value utilization of *Indigofera tinctoria* leaves.

[0004] This invention innovatively proposes a green and efficient biotransformation method to prepare a new indigo extract from *Strobilanthes cusia* using a self-made and low-cost endogenous enzyme. This method does not involve strong acids or toxic solvents, using only water as the transformation medium. This endogenous enzyme transformation method can rapidly produce high-quality indigo extract, while simultaneously employing membrane separation coupled with freeze-drying technology to rapidly treat fermentation wastewater. This not only recovers a higher-value-added phenolic acid extract but also enables water recycling. Therefore, the integrated production process of the above-mentioned *Strobilanthes cusia* indigo extract and phenolic acid extract is green and environmentally friendly. Furthermore, this invention systematically evaluates the staining effect, antibacterial activity, and wound healing ability of the *Strobilanthes cusia* indigo extract and phenolic acid extract, laying the foundation for the extension and sustainable healthy development of the *Strobilanthes cusia* industry chain. Summary of the Invention

[0005] In view of this, the present invention provides an integrated preparation method for indigo extract and phenolic acid extract.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: An integrated preparation method of indigo extract and phenolic acid extract, comprising the following steps: S1. Preparation of crude extract of endogenous enzymes from *Indigofera tinctoria* leaves Fresh leaves of *Indigofera tinctoria* were extracted by low-temperature water cell wall disruption, filtered, concentrated by microfiltration membrane, and freeze-dried to obtain crude extract of endogenous enzymes from *Indigofera tinctoria* leaves.

[0007] S2. Preparation of Indigofera tinctoria extract The raw material of *Indigofera tinctoria* is subjected to high-temperature enzyme inactivation, water extraction, enzyme conversion, alkali precipitation, separation, and drying to obtain *Indigofera tinctoria* extract.

[0008] Preparation of S3, physolic acid extract Wastewater generated during the preparation of Indigofera tinctoria extract is purified by microfiltration, concentrated by reverse osmosis, and dried to obtain Indigofera tinctoria extract.

[0009] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the cell wall breaking extraction in step S1 includes enzymatic cell wall breaking extraction, ultrasonic cell wall breaking extraction, mechanical cell wall breaking extraction, and free combinations of the above three cell wall breaking extraction methods.

[0010] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the indigo raw material in step S2 includes indigo leaves, indigo stems and leaves, indigo stalks and the whole indigo plant. Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the high-temperature enzyme inactivation in step S2 includes high-temperature steam inactivation, microwave inactivation, hot water blanching inactivation, and high-temperature drying inactivation.

[0011] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the water extraction in step S2 includes low-temperature water extraction, room-temperature water extraction and high-temperature water extraction.

[0012] Preferably, in the above-mentioned integrated preparation method of Indigofera tinctoria extract and phenolic acid extract, the enzyme in step S2 is characterized by being a crude extract of Indigofera tinctoria leaf endogenous enzymes, as well as all enzymes capable of breaking glycosidic bonds in indole glycosides, such as β-glucosidase.

[0013] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the alkali used in the alkali precipitation in step S2 includes all alkalis with strong alkalinity such as potassium hydroxide, sodium hydroxide, barium hydroxide, and calcium hydroxide.

[0014] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the separation method in step S2 includes disc centrifugation, plate and frame filtration, candle filtration, etc.

[0015] Preferably, in the above-mentioned integrated preparation method of indigo extract and phenolic acid extract, the drying in step S3 includes freeze drying and spray drying.

[0016] Preferably, the above-mentioned integrated preparation method of indigo extract and phenolic acid extract includes the following steps: S1. Preparation of crude extract of endogenous enzymes from *Indigofera tinctoria* leaves Weigh a certain amount of fresh *Indigofera tinctoria* leaves, add 50 mmol / L phosphate buffer solution (pH=6.0) at 0-10 ℃, and then perform cell wall disruption in a high-speed blender with a cold water circulation and heat preservation device for 0-30 min. After cell wall disruption, perform the first extraction in the high-speed blender at 0-10 ℃, with a solid-liquid ratio of 1:5-1:20 and an extraction time of 0.5-3 h. Filter, and add 50 mmol / L phosphate buffer solution (pH=6.0) at 0-10 ℃ to the filter residue for a second extraction, with a solid-liquid ratio of 1:2-1:10 and an extraction time of 10-60 min. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10-60 min to obtain a membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40 ℃; -40~-20 ℃, 3~9 h; -20~10 ℃, 2~8 h; 10~25 ℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0017] or Weigh a certain amount of fresh *Indigofera tinctoria* leaves and cut them into 0.5–5 cm pieces with a chaff cutter. Add 50 mmol / L (pH=6.0) phosphate buffer solution at 0–10 °C and place the mixture in an ultrasonic cleaner for the first extraction. The ultrasonic power is 0–100 W, the solid-liquid ratio is 1:5–1:20, and the extraction time is 10–60 min. Filter the mixture, and add 50 mmol / L (pH=6.0) phosphate buffer solution at 0–10 °C to the residue for the second extraction. The ultrasonic power is 0–100 W, the solid-liquid ratio is 1:2–1:10, and the extraction time is 5–30 min. Combine the two extracts to obtain the crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10–60 min to obtain the membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40 ℃; -40~-20 ℃, 3~9 h; -20~10 ℃, 2~8 h; 10~25 ℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0018] or Weigh a certain amount of fresh *Indigofera tinctoria* leaves and cut them into 0.5–5 cm pieces using a chaff cutter. Add 0–10 °C phosphate buffer solution (50 mmol / L, pH=6.0) and 0.1–0.5% cellulase or pectinase, then place the mixture in a constant temperature extraction apparatus for the first extraction at a solid-liquid ratio of 1:5–1:20 for 1–5 h. Filter the mixture, and add 0–10 °C phosphate buffer solution (50 mmol / L, pH=6.0) again to the filter residue for a second extraction at a solid-liquid ratio of 1:2–1:10 for 1–5 h. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10–60 min to obtain a membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40 ℃; -40~-20 ℃, 3~9 h; -20~10 ℃, 2~8 h; 10~25 ℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0019] or Weigh a certain amount of fresh *Indigofera tinctoria* leaves and cut them into 0.5–5 cm pieces with a chaff cutter. Add 0–10 °C phosphate buffer solution (50 mmol / L, pH=6.0) and 0.1–0.5% cellulase or pectinase, then place the mixture in an ultrasonic cleaner for the first extraction. The ultrasonic power is 0–100 W, the solid-liquid ratio is 1:5–1:20, and the extraction time is 10–60 min. Filter the mixture, and add 0–10 °C phosphate buffer solution (50 mmol / L, pH=6.0) again to the filter residue for a second extraction. The ultrasonic power is 0–100 W, the solid-liquid ratio is 1:2–1:10, and the extraction time is 5–30 min. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10–60 min to obtain a membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40℃; -40~-20℃, 3~9 h; -20~10℃, 2~8 h; 10~25℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0020] or Weigh a certain amount of fresh *Indigofera tinctoria* leaves, add 0-10℃ phosphate buffer solution (50 mmol / L, pH=6.0) and 0.1-0.5% cellulase or pectinase, and then use a high-speed blender with a cold water circulation and heat preservation device for 0-30 min to break down the cells. After the cell wall breaking process, perform the first extraction in the high-speed blender at 0-10℃, with a solid-liquid ratio of 1:5-1:20 and an extraction time of 0.5-3 h. Filter, and add 0-10℃ phosphate buffer solution (50 mmol / L, pH=6.0) to the filter residue for a second extraction, with a solid-liquid ratio of 1:2-1:10 and an extraction time of 10-60 min. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10-60 min to obtain a membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40 ℃; -40~-20 ℃, 3~9 h; -20~10 ℃, 2~8 h; 10~25 ℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0021] or Weigh a certain amount of fresh *Indigofera tinctoria* leaves, add 50 mmol / L phosphate buffer solution (pH=6.0) at 0-10℃, and then use a high-speed blender with a cold water circulation and heat preservation device for cell wall disruption for 0-30 min. After cell wall disruption, place the mixture in an ultrasonic cleaner for the first extraction, with an ultrasonic power of 0-100 W, a solid-liquid ratio of 1:5-1:20, and an extraction time of 10-60 min. Filter the mixture, and add 50 mmol / L phosphate buffer solution (pH=6.0) at 0-10℃ again for the second extraction, with an ultrasonic power of 0-100 W, a solid-liquid ratio of 1:2-1:10, and an extraction time of 5-30 min. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane for 10-60 min to obtain a membrane concentrate. The membrane concentrate was directly freeze-dried with the following specific process parameters: pre-freezing to -40 ℃; -40~-20 ℃, 3~9 h; -20~10 ℃, 2~8 h; 10~25 ℃, 0.5~5 h) to obtain crude extract of endogenous enzymes.

[0022] S2. Preparation of Indigofera tinctoria extract Place the leaves, stems, or stalks of *Isatis tinctoria* (cut into 5-10 cm sections) or the whole plant of *Isatis tinctoria* (cut into 10-20 cm sections) into a high-temperature steam cooker, spreading them to a thickness of 1-10 cm, and introduce steam for 5-60 minutes.

[0023] or Place the leaves, stems, or stalks of *Isatis tinctoria* (cut into 5-10 cm sections) or the whole plant of *Isatis tinctoria* (cut into 10-20 cm sections) in a microwave device with a dehumidifying function, set the power to 500-900 W, and the time to 5-30 min.

[0024] or Blanch the leaves, stems, or stalks of *Ipomoea purpurea* (cut into 5-10 cm sections) or the whole plant (cut into 10-20 cm sections) in hot water at 80-100℃ for 5-30 minutes, with a solid-liquid ratio of 1:5-10.

[0025] or Place the leaves, stems, or stalks of *Isatis tinctoria* (cut into 5-10 cm sections) or the whole plant of *Isatis tinctoria* (cut into 10-20 cm sections) into a dryer. Set the inlet temperature to 600-1100℃ and the outlet temperature to 60-120℃ for 5-30 minutes.

[0026] The enzyme-inactivated raw material of *Indigofera tinctoria* was extracted in low-temperature water at 0-10 ℃ with a solid-liquid ratio of 1:5-20, an extraction time of 0.5 h-5 h, and 1-5 extractions. After extraction, the extract was filtered to obtain the extract.

[0027] or The enzyme-inactivated raw material of *Indigofera tinctoria* was extracted in room temperature water at 10-30 ℃. The solid-liquid ratio was set to 1:5-20, the extraction time was 0.5 h-5 h, and the extraction was performed 1-5 times. After the extraction was completed, the extract was filtered to obtain the extract.

[0028] or The enzyme-inactivated raw material of *Indigofera tinctoria* was extracted in high-temperature water at 50-80 ℃. The solid-liquid ratio was set to 1:5-20, the extraction time was 0.5 h-5 h, and the extraction was performed 1-5 times. After the extraction was completed, the extract was filtered to obtain the extract.

[0029] When the temperature of the extract rises or falls to 30-50 ℃, add 0.1-1.0% of the crude endogenous enzyme extract (by weight of the raw material) and then place it in a constant temperature device at 30-70 ℃ for fermentation for 5-20 h.

[0030] or When the temperature of the extract rises or falls to 30-50 ℃, add 0.5-2.0‰ of β-glucosidase (based on the weight of the raw material) and adjust the pH to 5-7 with disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution. Then, place it in a constant temperature device at 30-70 ℃ for fermentation for 5-20 h.

[0031] After the enzyme fermentation is completed, the pH of the fermentation broth is adjusted to 8-11 with 5-20% sodium hydroxide solution, and then the reaction is carried out at 30-70 ℃ for 5-20 h.

[0032] or After the enzyme fermentation is completed, the pH of the fermentation broth is adjusted to 8-11 with 5-20% potassium hydroxide solution, and then the reaction is carried out at 30-70 ℃ for 5-20 h.

[0033] or After the enzyme fermentation is completed, the pH of the fermentation broth is adjusted to 8-11 with 5-20% calcium hydroxide solution, and then the reaction is carried out at 30-70 ℃ for 5-20 h.

[0034] or After the enzyme fermentation is completed, the pH of the fermentation broth is adjusted to 8-11 with 5-20% barium hydroxide solution, and then the reaction is carried out at 30-70 ℃ for 5-20 h.

[0035] After the reaction was complete, all the liquid was centrifuged using a disc centrifuge to obtain the centrifuged liquid and precipitate. The precipitate was dried in an oven at 50-70°C to obtain the indigo extract.

[0036] or After the reaction was complete, all the liquid was passed through a candle filter to obtain a filtrate and a filter cake. The filter cake was dried in an oven at 50-70 °C to obtain the indigo extract.

[0037] or After the reaction was complete, all the liquid was passed through a plate and frame filter press to obtain filtrate and filter cake. The filter cake was dried in an oven at 50-70 °C to obtain indigo extract.

[0038] Preparation of S3, physolic acid extract Apply a microfiltration membrane to the centrifuged liquid or filtrate, with a filtration area of ​​0.5–2.0 μm. 2 The time is 1-5 hours, and the average flux is 1000-3000 L / (m²). 2 (h) to obtain the clear liquid from the microfiltration membrane. The clear liquid from the microfiltration membrane is then concentrated using a reverse osmosis membrane with a filtration area of ​​0.5~2.0 m². 2 The time is 2–10 h, and the average flux is 500–2000 L / (m²). 2 The concentrate was obtained by freeze-drying the concentrate at -40℃ for 5-10 h; then at -40 to -20℃ for 5-10 h; then at -20 to 10℃ for 5-10 h; and finally at 10 to 25℃ for 0.5-5 h to obtain the phenolic acid extract of Astragalus membranaceus leaves.

[0039] or Apply a microfiltration membrane to the centrifuged liquid or filtrate, with a filtration area of ​​0.5–2.0 μm. 2 The time is 1-5 hours, and the average flux is 1000-3000 L / (m²). 2 (h) to obtain the clear liquid from the microfiltration membrane. The clear liquid from the microfiltration membrane is then concentrated using a reverse osmosis membrane with a filtration area of ​​0.5~2.0 m². 2 The time is 2–10 h, and the average flux is 500–2000 L / (m²). 2 The concentrated solution was obtained by spray drying. The specific process parameters were as follows: inlet air temperature 180~200 ℃, outlet air temperature 60~90 ℃, feed concentration 10~30%, feed flow rate 50~80 L / h, hot air velocity 0.5~3 m / s, and drying tower pressure -50~-200 Pa; *Indigofera tinctoria* leaf phenolic acid extract was obtained.

[0040] As can be seen from the above technical solution, compared with the prior art, this invention discloses an integrated preparation method for indigofera tinctoria extract and phenolic acid extract. This method is not only green and efficient, but also generates no wastewater during the entire production process. The indigofera tinctoria extract prepared using this method has superior dyeing effects compared to natural Indian indigo. Furthermore, the prepared indigofera tinctoria phenolic acid extract is mainly composed of active ingredients such as polyphenols, organic acids, and polysaccharides, exhibiting good broad-spectrum antibacterial activity and wound-healing ability. Therefore, the developed indigofera tinctoria extract and phenolic acid extract have good application potential in the fields of dyes, daily chemicals, and feed additives. Attached Figure Description

[0041] 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.

[0042] Figure 1 Preparation technology roadmap of Indigofera tinctoria extract and phenolic acid extract Figure 2 Dyeing effects of products with different indigo contents Figure 3 Images of inhibition zones of phenolic acid extracts against four types of bacteria Figure 4 Effects of different groups on wound healing in mice of different ages Figure 5 Trends in body weight (a), wound healing area (b), and wound healing rate (c) of mice in different groups. Detailed Implementation 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.

[0043] Example 1 Weigh 5 kg of fresh *Indigofera tinctoria* leaves and add 50 L of phosphate buffer solution (50 mmol / L, pH=6.0) at 4 ℃. Then, use a high-speed blender with a cold water circulation and insulation device to break down the cells for 10 min. After the cell-wall breaking process, continue extraction in the blender at 4 ℃ for 1 h. Filter the mixture, and add 20 L of phosphate buffer solution (50 mmol / L, pH=6.0) at 4 ℃ for a second extraction of 30 min. Combine the two extracts to obtain a crude enzyme extract. Treat the crude enzyme extract with a microfiltration membrane to obtain a permeate and a concentrate. Freeze-dry the concentrate directly (specific process parameters are as follows: pre-freeze to -40 ℃; -40~-20 ℃, 6 h; -20~10 ℃, 5 h; 10~25 ℃, 1 h) to obtain the crude endogenous enzyme extract.

[0044] Example 2 100 kg of fresh *Indigofera tinctoria* leaves were weighed and then divided into 5 batches, each batch placed in 500 L of boiling water for enzyme inactivation. The enzyme-inactivated leaves were then placed in an extraction tank containing 800 L of hot water at 80 °C for 1.5 h. After extraction, the mixture was filtered, and 700 L of water at 40 °C was added to the residue for a second extraction for 1.0 h. After extraction, the residue was washed with an appropriate amount of room temperature water. The blanching liquid, the two extractions, and the washing liquid were combined. All liquids were piped and pumped into a fermenter, and 500 g of crude endogenous enzyme extract was added before fermentation for 24 h. After fermentation, NaOH solution was added to the fermentation broth to adjust the pH to approximately 9.5, and the reaction continued for 6 h. After the reaction, all liquids were filtered through a disc filter to obtain filtrate and filter cake. The filter cake was dried in a 60 °C oven to obtain indigo extract, containing 40.2% indigo and 2.03% indirubin.

[0045] Example 3 The filtrate was filtered through a microfiltration membrane with a filtration area of ​​1.5 m². 2 The time was 1 hour, and the average flux was 2000 L / (m³). 2 (h) to obtain the clear solution from the microfiltration membrane. The clear solution from the microfiltration membrane is then concentrated using a reverse osmosis membrane with a filtration area of ​​1.0 m². 2 The time was 2 hours, and the average flux was 1000 L / (m³). 2The concentrate was obtained by freeze-drying. The specific process parameters were as follows: pre-freezing to -40 ℃; -40~-20 ℃ for 8 h; -20~10 ℃ for 7 h; 10~25 ℃ for 2 h; to obtain the phenolic acid extract of *Indigofera tinctoria* leaves. The polysaccharide content, organic acid content, flavonoid content, and polyphenol content were determined to be 10.1%, 7.3%, 3.56%, and 5.32% respectively, using the Kjeldahl method, weight loss method, anthrone-sulfuric acid method, and Folin-Ciocalteu colorimetric method.

[0046] Example 4 Samples of indigo extract (40.2% indigo content, NIE), natural Indian indigo (20% indigo content, YIE-1), natural Indian indigo (40% indigo content, YIE-2), Changzhou University indigo extract (30% indigo content, CIE), and synthetic indigo (99.8% indigo content, SI) were weighed separately and thoroughly shaken and dissolved in a room temperature shake dyeing tester. Penetrant DM-1228 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 penetrant DM-1228 (two dips and two nips), then dipped and nipped 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.

[0047] The dyeing effects of products with different indigo contents, such as Figure 2 As shown. 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. The larger the K / S value, the better the dyeing performance. Calculations show that the K / S value of NIE is 11.7, which is greater than SI (11.2), YIE-1 (10.0), YIE-2 (8.78), and CIE (9.63). Furthermore, the wash fastness, dry rubbing fastness, and wet rubbing fastness of NIE are all between 4 and 5. Therefore, the prepared indigo extract has a superior dyeing effect compared to synthetic indigo and commercially available natural indigo extracts.

[0048] Example 5 An appropriate amount of phenolic acid extract was taken and its inhibitory activity against *Escherichia coli*, *Staphylococcus aureus*, *Bacillus cereus*, and *Pseudomonas aeruginosa* was analyzed. First, appropriate amounts of bacteria from each of the four bacterial strains were inoculated into 100 mL of LB liquid medium and then placed in a constant temperature shaker (30 ℃) for overnight activation. The revived bacterial suspensions were then diluted with sterile PBS to a concentration of 1.6 × 10⁻⁶. 8 CFU / mL, then 100 μL was evenly spread onto LB solid medium. Wells were punched using a 10 mm punch, and 100 μL of sterile sample solutions of different concentrations were added to each well. An equal volume of sterile water was added to the control group, and the plates were incubated at 37°C for 24 h. Sterile water and gentamicin sulfate (GS) were used as blank and sample controls, respectively. After the incubation process, images of the inhibition zones were taken using a regular camera. 100 μL of LB solid medium was transferred to each well of a 96-well microtiter plate, followed by 100 μL of phenolic acid extract (2.0 mg / mL) and GS (1.0 mg / mL) solutions in the first row of microtiter plates. These solutions were diluted twice with nutrient broth. Finally, 100 μL of *Escherichia coli*, *Staphylococcus aureus*, *Bacillus cereus*, or *Pseudomonas aeruginosa* was added to each well. All microtiter plates were incubated at 30°C for 24 h, and the MIC values ​​were obtained.

[0049] Images of the inhibition zones of phenolic acid extracts against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli are shown below. Figure 3 As shown, at the same concentration, the inhibitory effects of the phenolic acid extract on four bacteria were as follows: Staphylococcus aureus > Pseudomonas aeruginosa > Bacillus cereus > Escherichia coli. Among these bacteria, the phenolic acid extract showed the best inhibitory effect on Staphylococcus aureus, with a MIC value of 32 μg / mL, close to that of gentamicin sulfate. Therefore, the prepared phenolic acid extract possesses good broad-spectrum antibacterial activity and will show great application potential in the fields of daily chemicals, pharmaceuticals, and feed additives. The phenolic acid extract is composed of abundant polysaccharides, organic acids, flavonoids, and polyphenols, all of which have excellent antibacterial capabilities, which may be the main reason for its superior antibacterial activity.

[0050] Example 6 Female mice were acclimatized for one week. The rearing process was conducted in a barrier system laboratory (air cleanliness ≤10000, ventilation rate 10-20 times / h, temperature 20-26 ℃, daily temperature difference ≤3 ℃, relative humidity 40-70%), with 12 hours of light and 12 hours of dark circulation, and adequate food and water provided. After anesthetizing the mice with isoflurane, their backs were shaved and disinfected with 75% alcohol. Then, using surgical scissors, a circular full-thickness incision with a diameter of 8 mm was created on the back of each mouse, penetrating deep into the fascia. 10 μL of 1x106 A CFU / mL suspension of Staphylococcus aureus was uniformly dispersed on the wound and sealed with a 3M film. Twenty-four hours after infection, turbid pus appeared on the wound, indicating successful infection modeling. The modeling day was designated D0, and the day after infection completion was designated D1.

[0051] Phosphate-buffered saline (PBS), penicillin solution (PCN, 64 μg / mL), and phenolic acid extract solution (128 μg / mL) were irradiated under UV light for 30 min for later use. 40 μL of PBS, PCN, and phenolic acid extract solution were uniformly applied to the wounds of mice, and the wounds were sealed with 3M Tegarderm Film transparent dressings on days 1, 3, 5, 7, 11, and 14. All mice were weighed, wound photographs were taken, and the healing rate was calculated on days 1, 3, 5, 7, 11, and 14. The wound healing rate was calculated using the following formula: Wound healing rate (%) = (S1 - Sn) / S1 × 100%, where S1 is the wound area (cm²) on day 1. 2 Sn represents the wound area (cm) on a specific day. 2 ).

[0052] The trends of body weight, wound healing area, and wound healing rate in mice of different groups are as follows: Figure 5 As shown. From Figure 5 As can be seen, the body weight of each group of mice increased over time, indicating that the phenolic acid extract had no toxicity or side effects on the mice. As the experiment progressed, all experimental groups showed significant therapeutic effects in wound healing. Particularly noteworthy is that from day 3 of the experiment, the wound healing rate of each experimental group showed a significant upward trend, with statistically significant differences (p<0.05). At the end of the experiment, the wound healing rate of the phenolic acid extract exceeded 98%. In conclusion, by analyzing wound healing indicators and mouse body weight, the excellent ability of the phenolic acid extract to promote wound healing has been clearly demonstrated, laying the foundation for its subsequent application in the fields of daily chemicals and pharmaceuticals.

[0053] 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.

[0054] 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. An integrated preparation method of indigo extract and phenolic acid extract, characterized in that... Includes the following steps: S1. Preparation of crude extract of endogenous enzymes from *Indigofera tinctoria* leaves Fresh leaves of *Indigofera tinctoria* were subjected to low-temperature water cell wall disruption extraction, filtration, membrane concentration, and freeze-drying to obtain a crude extract of endogenous enzymes from *Indigofera tinctoria* leaves. S2. Preparation of Indigofera tinctoria extract The raw material of *Indigofera tinctoria* is subjected to high-temperature enzyme inactivation, water extraction, enzyme conversion, alkali precipitation, separation, and drying to obtain *Indigofera tinctoria* extract; Preparation of S3, physolic acid extract The wastewater generated during the preparation of Indigofera tinctoria extract is purified by membrane removal, concentrated, and dried to obtain Indigofera tinctoria extract.

2. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The cell wall disruption extraction described in step S1 includes enzymatic cell wall disruption extraction, ultrasonic cell wall disruption extraction, mechanical cell wall disruption extraction, and free combinations of the above three cell wall disruption extraction methods.

3. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The raw materials for strychnifolia mentioned in step S2 include strychnifolia leaves, strychnifolia stems and leaves, strychnifolia stalks and whole strychnifolia plants.

4. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The high-temperature enzyme inactivation method mentioned in step S2 includes high-temperature steam inactivation, microwave inactivation, hot water blanching inactivation, and high-temperature rapid drying inactivation.

5. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The water extraction described in step S2 includes low-temperature water extraction, room-temperature water extraction, and high-temperature water extraction.

6. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The enzymes mentioned in step S2 are crude extracts of endogenous enzymes from *Indigofera tinctoria* leaves, as well as all enzymes capable of breaking glycosidic bonds in indole glycosides, such as β-glucosidase.

7. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The alkali used in the alkali precipitation in step S2 includes all alkalis with strong alkalinity, such as potassium hydroxide, sodium hydroxide, barium hydroxide, and calcium hydroxide.

8. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The separation methods described in step S2 include disc centrifugation, plate and frame filtration, candle filtration, etc.

9. The integrated preparation method of indigo extract and phenolic acid extract according to claim 1, characterized in that, The drying process described in step S3 includes freeze drying and spray drying.

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