Method for preparing multifunctional indigo and polyphenol extract from malan stem and leaf

Abstract

Using the stems and leaves of *Indigofera tinctoria* as raw material, a combined high-pressure water extraction technology involving water spray washing and grading, along with steam explosion, was employed to effectively improve the release rate of active substances such as indole glycosides and polyphenols, as well as the conversion rate of indigo. Multifunctional indigo and polyphenol extracts were prepared through bio-fermentation and membrane fractionation purification. The indigo extract contained significantly more bioactive substances than traditional lime-based indigo powder and indigo acidified compounds, with 15 times more indigo, 25 times less ash, and 650 times less calcium. Compared to natural Indian indigo, it exhibited superior dyeing performance on cotton fabrics and showed better inhibitory effects against *Aspergillus niger*, *Staphylococcus aureus*, and *Escherichia coli*. The *Indigofera tinctoria* polyphenol extract, composed of phenolic acids, phenylethyl glycosides, flavonoids, and polysaccharides, demonstrated excellent inhibitory activity against *Staphylococcus aureus* (IC50 = 32 μg / mL) and a wound healing rate exceeding 98% in mice, exhibiting good broad-spectrum antibacterial and wound-healing properties.

Description

Technical Field

[0001] This invention belongs to the field of dye, daily chemical and functional feed additive development, and specifically relates to a method for preparing multifunctional indigo and polyphenol extracts from the stems and leaves of Indigofera tinctoria. Background Technology

[0002] Indigofera tinctoria is a perennial herb native to southeastern and southwestern China, India, Japan, and the Indochina Peninsula. Its stems and leaves are rich in indole glycosides and other indigo precursors, as well as polyphenolic active ingredients. Polyphenols, in particular, possess anti-inflammatory, antibacterial, antioxidant, anti-tumor, whitening, anti-glycation, and astringent properties, and are widely used in biomedicine, functional foods, skincare, and antibiotic-alternative feeds. Indole glycosides are mainly distributed in the vacuoles of the leaf or stem cells of Indigofera tinctoria, coexisting with the highly active β-glucosidase in the plant's chloroplasts. β-glucosidase hydrolyzes the glycosidic bonds of indole glycosides in fresh leaves, releasing 3-hydroxyindolephenol, which is chemically extremely unstable and reacts rapidly with oxygen, spontaneously oxidizing to form indolequinone or indigo, with a very small portion further oxidizing and dimerizing to form indigo, indirubin, etc. Historically, Indigofera tinctoria has been used to prepare natural indigo dye and cinnabar. The traditional processing mainly involves converting precursors such as indoleglycosides and indorubane B from the leaves or stems of scotch, indigofera, and isatis indigotica into active ingredients like indigo and indirubin. Lime plays a crucial role in this process; the alkaline environment it provides allows indolephenol to undergo a condensation oxidation reaction, producing indigo and indirubin. However, indigo and indirubin are light and poorly soluble in water, making them difficult to collect. Lime (which reacts with water to form Ca(OH)2) and the CaCO3 produced by reacting with the soaking solution can act as a carrier, allowing indigo and indirubin to precipitate and form indigo paste. The indigo paste is then dried into indigo powder or purified to produce indigo powder, thus forming the traditional lime-based indigo processing technology. Because the production of indigo paste using lime (generally 5-10% of the fresh leaf weight) uses a large amount of lime, the resulting indigo product contains a large amount of inorganic substances such as CaCO3, Ca(OH)2, and SiO2, with a total ash content of 60-88%. The pH of the sample water layer is also high. The presence of large amounts of lime and other inorganic substances not only seriously affects the clinical efficacy of indigo but also has a strong irritant effect on the digestive tract. In addition, the large amount of alkaline wastewater generated during production significantly increases wastewater treatment costs. Therefore, developing more environmentally friendly production processes and higher value-added indigo extracts is urgently needed. Indigo extract is produced directly from the fermentation of *Indigofera tinctoria* without the addition of lime to form indigo. Compared to indigo paste and cinnamon, indigo extract contains dozens of times more functional active ingredients and has properties similar to both biomedicine and high-quality plant dyes, making its applications more extensive. Furthermore, as the most important natural blue dye, indigo extract, due to its lime-free nature and indigo content exceeding 40%, 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, with better lightfastness and wash resistance. However, research on indigo extract in my country is relatively limited, and there are no precedents for the industrial-scale production of indigo without lime. Zheng Jin et al. developed a highly efficient process for producing indigo extract from *Indigofera tinctoria* 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 innovative work on indigo extract, inventing chemical and exogenous enzymatic conversion methods. For example, Zhang Changwei 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 Hongxia et al. prepared an indigo extract with an indigo content of 37.4% and an indirubin content of 2.30% using an exogenous enzyme conversion method, and found that the prepared indigo extract maintained the same chemical composition as conventional indigo. Steam explosion technology is an emerging physical modification technique. Its main principle involves placing the material in a high-temperature, high-pressure, sealed container—a steam explosion machine. The raw material is swelled by superheated liquid, and steam penetrates into the plant tissue. When the high pressure is instantaneously released (within 0.00875 s), the pressure difference causes the high-temperature liquid in the pores of the raw material to rapidly vaporize and expand, leading to cell "explosion." The instantaneous mechanical action, high temperature and pressure, and rapid expansion of steam during steam explosion can disrupt the interconnections between cellulose, hemicellulose, and lignin, changing them from a rigid, ordered state to a rough, disordered state. This breaks down the natural barriers formed during plant growth, facilitating the dissolution of active substances. Simultaneously, it breaks hydrogen bonds, glycosidic bonds, ether bonds, and ester bonds connected to polyphenols and glycosides, resulting in the transfer and transformation of active ingredients. Steam explosion and hydrothermal extraction are beneficial for the release of active substances such as indole glycosides and polyphenols, thereby increasing the extraction rate. This invention innovatively proposes a method for preparing multifunctional indigo and polyphenol extracts from the stems and leaves of *Indigofera tinctoria*. This method does not involve strong acids or toxic solvents, and only uses water as the conversion medium. It can quickly prepare high-quality indigo extracts and polyphenol extracts, and realize the recycling of wastewater in the processing of *Indigofera tinctoria*. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing multifunctional indigo and polyphenol extracts from the stems and leaves of *Indigofera tinctoria*. To achieve the above objective, the present invention adopts the following technical solution, specifically including 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 10-80 cm on the same day, cut off 20-50 cm of woody stems with a hydraulic cutter, retain 10-30 cm of tender branches and leaves, and spray them evenly with water with pH 4-7 to remove dirt and other impurities. The second step is steam explosion hydrothermal extraction: the washed Stellera chamaejasminoides raw material is added to a steam explosion container, water with pH 4~7 is added, the volume ratio of Stellera chamaejasminoides raw material to water (g / mL) is 1:20~60, the steam pressure of the container reaches 0.5~2.5 MPa, the heat preservation time is 0.5~10 min, and then the pressure is released and the temperature is lowered to 20~50℃. The third step is solid-slag separation: the steam explosion hydrothermal extract is passed through a 10-40 mesh stainless steel screen, the filter residue is cleaned with tap water, and the filtrate is combined. Step 4, biological fermentation: Add 0.01~6% biological enzymes to the filtrate from step 3 for fermentation, with a fermentation pH of 5~10, a temperature of 30~60℃, and a time of 8~72 h; Step 5, membrane fractionation purification of indigo extract: The bio-fermentation broth from step 4 is clarified by passing it through a microfiltration membrane. The volume ratio of permeate to concentrate is 3~12:1. The concentrate is separated by centrifugation. The centrifuged precipitate is dried to obtain indigo extract with an indigo content greater than 40%, ash content less than 2%, calcium content less than 0.4 g / kg, lead (Pb) less than 10 mg / kg, and arsenic (As) less than 10 mg / kg. Step 6, Preparation of polyphenol extract: Combine the permeate from the microfiltration membrane in step 5 and the supernatant separated by centrifugation. Remove impurities by ultrafiltration membrane with a molecular weight of 10,000 to 50,000 Daltons. The volume ratio of permeate to concentrate is 3 to 10:1. The permeate is then separated by nanofiltration membrane with a volume ratio of 3 to 6:1. The concentrate is dried by microwave vacuum to obtain polyphenol extract with a total polyphenol content of more than 20%, of which chlorogenic acid is more than 5% and polysaccharides are more than 10%. This patent reveals that the indigo precursor content in fresh leaves of *Indigofera tinctoria* is 5-10 times higher than that in the woody stems. Therefore, the selected tender leaves and branches of *Indigofera tinctoria* have a moisture content of 70-85% and an indole glycoside content of 0.5-2.5%, while the woody stems have a moisture content of 70-80% and an indole glycoside content of only 0.02-0.1%. Fresh *Indigofera tinctoria* contains indole glycoside alkaloids, which, under the action of glycosidases, lose glucose to exist in the form of indolephenol. Indolephenol is unstable; when it is slightly oxidized, for example, when exposed to air, two indoleoxy groups oxidize and combine to form indigo (blue). In traditional lime-based indigo paste production, the raw material for *Indigofera tinctoria* (also known as storax) is typically soaked in a soaking tank along with the stems and tender leaves. This process is not only bulky but also results in low conversion rates, low indigo content, and significant wastewater and waste residue, causing environmental pollution. To address these issues, this patent employs a grading process for fresh *Indigofera tinctoria* raw materials. Fresh stems and leaves, harvested on the same day (10-80 cm), are cut with a hydraulic cutter, removing 20-50 cm of woody stems and retaining 10-30 cm of tender leaves, preferably 10-20 cm from the leaf area. Indole glycosides are located in the vacuoles of *Indigofera tinctoria* tissue, while glucosidase, which degrades indole glycosides, resides in the mitochondria. During grading, the tissue is damaged, causing the glucosidase and indole glycosides to meet and degrade. Since indole glycosides are stable in a weakly acidic environment, this invention uses a pH 4-7 water spray for uniform washing, which not only removes impurities such as dirt but also maintains the stability of indole glycosides in the fresh leaves. Furthermore, in steps 1 and 2 above, the pH value adjustment is carried out using one of citric acid, acetic acid, oxalic acid, or sodium dihydrogen phosphate to adjust the pH of the aqueous solution to 5-7, with citric acid and acetic acid preferred for adjusting the pH of the aqueous solution to 5-6. Furthermore, this invention employs steam explosion hydrothermal extraction of *Indigofera tinctoria* raw material. The volume ratio (g / mL) of *Indigofera tinctoria* raw material to water is 1:20~60, preferably 1:20~30. The steam pressure reaches 0.5~2.5 MPa, preferably 0.5~1.5 MPa, and the holding time is 0.5~10 min. The active substances in *Indigofera tinctoria* stems and leaves, such as indole glycosides, polyphenols, organic acids, polysaccharides, and flavonoids, exist in a bound form, especially with cellulose and lignin. The active substances are encapsulated by cellulose and lignin, resulting in a low release rate. This patent utilizes hydrothermal high-temperature extraction at above 160℃ under acidic conditions (pH 5~6) in the aqueous solution, which not only kills microorganisms and indole glycosidases that degrade indole glycosides in fresh *Indigofera tinctoria* raw material, but also significantly improves the conversion rate of indigo. Furthermore, the bio-enzyme described in this invention is an enzyme capable of breaking the glycosidic bonds in indole glycosides, including any one or a combination of the endogenous enzyme extract from *Indigofera tinctoria* leaves, *Aspergillus niger* enzyme, *Cephalosporium* cellulase, and β-glucosidase. The endogenous enzyme extract and β-glucosidase are preferred. Enzymatic fermentation is carried out at a pH of 5-10, preferably 5-8, a temperature of 30-60°C, preferably 50-60°C, and a time of 8-72 h, preferably 12-24 h. The endogenous enzyme extract from *Indigofera tinctoria* leaves described in this invention is obtained by mechanically stirring and breaking down the cell walls of fresh *Indigofera tinctoria* leaves with a phosphate buffer solution at pH 5-6 and ice water, at a stirring speed of 3000-5000 r / min, a solid-liquid ratio of 1:2-5, and an extraction time of 0.5-1 h. The pulp residue is then directly freeze-dried to obtain a crude extract of the endogenous enzyme from *Indigofera tinctoria* leaves. During the bio-enzyme fermentation process, 0.01-6% enzyme is added to the filtrate, preferably 0.01-0.5% of exogenous enzyme and 0.5-6% of crude endogenous enzyme extract, preferably 0.5-6%. This patent employs microfiltration membrane fractionation to purify indigo extract, selecting any one of the following: a ceramic membrane with a pore size of 50-200 nm, a nano-polymer organic membrane, or a 0.3-50 μm stainless steel filter element, with a preferred pore size of 50-200 nm ceramic membrane. After clarification via the ceramic membrane, the permeate to concentrate volume ratio is 3-12:1, preferably 8-10:1. The concentrate is separated by centrifugation, and the centrifuged precipitate is dried in a vacuum drying oven at 60℃ and pulverized to obtain the indigo extract, wherein the indigo content is greater than 40%, the staining strength (of the standard) is 100 points, there are no harmful aromatic amines, the calcium content is less than 0.4 g / kg, the lead (Pb) content is less than 10 mg / kg, and the arsenic (As) content is less than 10 mg / kg. The quality of the indigo extract produced by this patent is significantly higher than that of traditional lime-based indigo powder, indigo, and its acidified derivatives. Typically, indigo powder contains more than 50% ash and more than 10% acid-insoluble ash, while indigo acidified derivatives contain more than 10% ash and more than 5% acid-insoluble ash. The indigo extract produced by this patent has less than 2% ash and only 1.59% acid-insoluble ash, indicating that the main components of the high-purity indigo sample are organic, representing a 25-fold reduction in ash compared to indigo powder. Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to evaluate the Ca content in indigo powder and indigo extract. The highest Ca content was found in indigo powder (27.5%), while the Ca content in indigo acidified derivatives decreased to approximately 0.3%, indicating that acidification significantly removes Ca ions. The Ca content in the high-purity indigo extract was only 0.38 g / kg (0.038%), a 650-fold reduction compared to indigo powder. The contents of As, Hg, and Pb were all below 10 mg / kg. Py-GC-MS analysis showed significant differences in the organic composition of the pyrolysis gas chromatography (280~600℃) analysis between traditionally processed indigo and the indigo extract of this patent. Two compounds were identified in the indigo paste powder sample, 12 compounds were identified in the indigo acid compound, and 19 compounds were identified in the indigo extract sample. The chemical composition of indigo powder, indigo acid, and indigo extract was further analyzed using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS). By comparing with the NIST 08 mass spectrometry database and using the area normalization method, the relative content of each component was obtained. Two organic compounds were identified from the indigo powder, 60 compounds from the indigo acid, and 184 compounds from the indigo extract. These compounds mainly include nine categories of substances: acids, polysaccharides, flavonoids, alkaloids, glycosides, polyphenols, aldehydes, proteins, and esters. This indicates that the indigo extract prepared by this invention contains more bioactive substances than the indigo powder and indigo acid obtained by traditional lime-based indigo production. This patent also found through antibacterial experiments that the inhibitory effect on Aspergillus niger, Staphylococcus aureus, and Escherichia coli all increased with increasing indigo content. Antibacterial activity: Indigo extract > Indigo acid compound > Indigo powder. High-content natural indigo extract has a good inhibitory effect on Aspergillus niger, with a MIC value of 0.5~1 μg / mL and an MBC value of 2~4 μg / mL. The indigo extract of this invention has excellent dyeing effect, with a dyeing strength (of standard) of 100 points, no harmful aromatic amines, a K / S value greater than 20, and dyeing performance and fastness on cotton fabrics of dyeing depth grade 1, washing fastness grade 3~4, perspiration fastness grade 3~5, dry rubbing fastness grade 3~5, and water fastness grade 3~5. Furthermore, this invention employs ultrafiltration and nanofiltration membranes for fractional purification to prepare polyphenol extracts. Impurities are removed using an ultrafiltration membrane with a molecular weight of 10,000 to 50,000 Daltons (preferably 10,000 to 30,000 Daltons). The volume ratio of the ultrafiltration permeate to the concentrate is controlled at 3 to 10:1, preferably 8 to 10:1. The ultrafiltration permeate is then separated using a nanofiltration membrane with a volume ratio of 3 to 6:1. The concentrate is then microwave-vacuum dried to obtain the polyphenol extract, containing more than 20% total polyphenols, of which more than 5% chlorogenic acid and more than 10% polysaccharides. Furthermore, this patent utilizes HPLC-IT-TOF-MS 2The chemical composition of polyphenols in *Indigofera tinctoria* leaves and polyphenol extracts was analyzed. The identification process included comparative analysis of retention time HPLC (RT) and mass spectrometry data with standards or literature reports, as well as comparisons with MassBank (https: / / bank.eu / MassBank / ) and HMDB (https: / / hmdb.ca / ) databases. For the first time, nine major phenolic substances were systematically identified, including at least three phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid), two phenylethanol glycosides (verbascoside, isoacteoside), and four flavonoids (isorhamnetin-3-O-glucoside, homoplantaginin, isorhamnetin, and hispidulin). Two distinct indole alkaloids (indole and indole glycoside) were also found. This patent further reveals that *Indigofera tinctoria* polyphenol extract possesses antibacterial activity and wound-healing ability, exhibiting good antibacterial activity against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Bacillus cereus*, and *Escherichia coli*, with MIC values ​​less than 45 mg / L. Furthermore, the inhibitory activity against Staphylococcus aureus was highest in Staphylococcus aureus, followed by Pseudomonas aeruginosa, then Bacillus cereus, and finally Escherichia coli. The MIC values ​​against Staphylococcus aureus and Escherichia coli were 21.88 and 43.75 mg / L, respectively, significantly lower than the positive control drug gentamicin sulfate (43.75 and 87.50 mg / L), indicating the best inhibitory effect against Staphylococcus aureus. The polyphenol extract showed no toxic side effects in mice and achieved a wound healing rate exceeding 98%. This clearly demonstrates the excellent ability of the polyphenol extract to promote wound healing. Therefore, the polyphenol extract possesses good broad-spectrum antibacterial activity and shows great application potential in the fields of daily chemicals, pharmaceuticals, and feed additives. The present invention has the following advantages: Compared to traditional lime-based indigo production, which results in low conversion rates and low indigo content, this invention utilizes a pH 4-7 water spray washing and grading process on fresh *Indigofera tinctoria* raw materials. This process not only removes impurities such as soil but also maintains the stability of indole glycosides in the fresh leaves. This patent reveals that the indigo precursor content is significantly higher than that of traditional *Indigofera tinctoria* woody stems by 5-10 times, and the indigo yield is increased by 2-3 times. Furthermore, this method generates no waste during production and recovers polyphenol extract byproducts, improving the utilization rate of *Indigofera tinctoria* resources. This invention employs steam explosion combined with high-pressure water extraction at pH 4-7 to effectively disrupt the interconnections between cellulose, hemicellulose, and lignin, thereby increasing the release rate of active substances such as indole glycosides, polyphenols, organic acids, polysaccharides, and flavonoids from the stems and leaves of *Indigofera tinctoria*. The hydrothermal high-temperature extraction under acidic conditions not only kills microorganisms and indole glycosidases in fresh *Indigofera tinctoria* raw materials but also significantly improves the conversion rate of indigo. The indigo extract obtained by this invention has an indigo content greater than 40%. It not only contains more bioactive substances than traditional lime-based indigo powder and indigo acidified products, but also exhibits a 15-fold increase in indigo content, a 25-fold reduction in ash content, and a 650-fold decrease in calcium content compared to indigo powder. Furthermore, it demonstrates significantly stronger inhibitory effects against Aspergillus niger, Staphylococcus aureus, and Escherichia coli. Compared to natural Indian indigo, it exhibits superior dyeing performance on cotton fabrics. Moreover, the prepared indigo polyphenol extract is mainly composed of polyphenols, flavonoids, organic acids, and polysaccharides, possessing good broad-spectrum antibacterial activity and wound-healing properties. Attached Figure Description 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. Figure 1 Evaluation of the antibacterial function of Indigofera tinctoria extract Figure 2 Evaluation of the antibacterial function of broccoli polyphenol extract Figure 3 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. Example 1: Preparation and quality analysis of Indigofera tinctoria extract Fresh *Indigofera tinctoria* stems and leaves, 10-80 cm long, harvested that day are used. 20-50 cm of the woody stems are removed using a cutting machine, leaving 10-30 cm of tender branches and leaves. The stems are then evenly sprayed with pH 5 water to remove dirt and other impurities. The washed *Indigofera tinctoria* stems are then added to a steam explosion container with pH 5.5 water. The volume ratio of *Indigofera tinctoria* stems to water (g / mL) is 1:20-60, preferably 1:40. The steam pressure in the container reaches 0.5-2.5 MPa, preferably 1.0-1.5 MPa, and the temperature is maintained for 0.5-10 min, preferably 5-10 min. The container is then depressurized and cooled to room temperature. The mixture is passed through a 10-40 mesh stainless steel sieve, and the residue is cleaned with tap water. The filtrates are combined. 0.1-6% of a biological enzyme is added to the filtrate for fermentation. The fermentation pH is 5-10, the temperature is 30-60℃, preferably 30-40℃, and the fermentation time is 8-72 h, preferably 8-12 h. h; then the fermentation broth is clarified through a microfiltration membrane, preferably an inorganic ceramic membrane, with a permeate to concentrate volume ratio of 3~12:1. The concentrate is separated by centrifugation, and the centrifuged precipitate is dried to obtain indigo extract with an indigo content greater than 40%, ash content less than 2%, calcium content less than 0.4 g / kg, lead (Pb) less than 10 mg / kg, and arsenic (As) less than 10 mg / kg. Example 2 Preparation and component analysis of bromosin polyphenol extract The permeate from the microfiltration membrane and the supernatant separated by centrifugation in Example 1 were combined and purified by ultrafiltration with a molecular weight of 10,000 to 50,000 Daltons (preferably 10,000 Daltons). The permeate-to-concentrate volume ratio was 3-10:1. The ultrafiltration permeate was then separated by nanofiltration with a permeate-to-concentrate volume ratio of 3-6:1. The concentrate was microwave-dried under vacuum to obtain a strychnine polyphenol extract with a total polyphenol content greater than 20% and chlorogenic acid greater than 5%. The extract was analyzed by HPLC-IT-TOF-MS. 2 The technology identified nine major compounds in polyphenol extracts using both positive and negative modes, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, verbascoside, isoverascoside, isokaempferol-3-O-glucoside, kaempferol, isorhamnetin, and kaempferol. Example 3 Evaluation of staining with Indigofera tinctoria extract The K / S value of indigo extract was tested using a computer colorimeter; the dyeing strength and dyeing depth of indigo extract were determined according to GB / T 2374; the water fastness of indigo extract was tested according to ISO 105-C10:2006; the rubbing fastness of indigo extract was analyzed using an electric rubbing fastness tester according to ISO 105-X12:2016; the washing fastness of indigo extract was tested according to AATCC 61-2013(R2020); and the perspiration fastness of indigo extract was tested based on KS K ISO 105E04:2010. The results showed that the dyeing strength (of the standard) of indigo extract was 100 points, the K / S value was greater than 20, and the dyeing performance and fastness on cotton fabrics were grade 1 for dyeing depth, grade 3-4 for washing fastness, grade 3-5 for perspiration fastness, grade 3-5 for dry rubbing fastness, and grade 3-5 for water fastness. Therefore, the indigo extract prepared by this invention has a superior dyeing effect compared to synthetic indigo and commercially available natural indigo extract. Example 4: Physicochemical Properties Analysis of Indigo Extraction 4.1 Experimental Samples Indigo paste powder and indigo paste acid are traditional indigo-making products, commercially available. The indigo extract in this patent is a self-made sample. 4.2 Ash Content Analysis Analysis was performed according to GB / T 21876. Approximately 3 g each of indigo paste powder, indigo acid compound, and indigo extract were accurately weighed and placed in a precisely weighed crucible that had been ignited to constant weight. The crucible was then placed in a box furnace and gradually heated to 600℃, followed by further ignition for 6 hours. The ash content of the indigo paste powder, indigo acid compound, and indigo extract was calculated based on the mass of the residue. 4.3 Determination of Acid-Insoluble Ash The analysis was performed according to GB 5009.4. 20 mL of 20% dilute hydrochloric acid was added to the crucible containing the ash residue from the above steps. The surface was covered with a watch glass and heated in an 80°C water bath for 10 min. The watch glass and crucible were washed with hot water and filtered through ashless filter paper. The filter paper and residue were then returned to the crucible and ignited until constant weight. Based on the mass of the residue, the acid-insoluble ash content of the test sample was calculated. 4.4 Thermogravimetric Analysis Thermogravimetric analysis (TGA) of the samples was performed using a 209F 1E-0090-L thermogravimetric analyzer. 5–10 mg of sample was placed in an Al₂O₃ crucible, and the temperature was programmed while the sample weight was recorded. The atmosphere during the test was N₂. The test temperature range was 40℃ to 600℃, and the programmed temperature increase was 10℃ / min. Thermogravimetric analysis was performed on indigo extract powder, indigo acid, indigo extract, and indigo standards. 4.5 Py-GC-MS Analysis This experiment used a PY-3030D / 7890B-5977A thermal decomposition chromatography-mass spectrometry (Py-GC-MS) system to analyze indigo extract powder, indigo acid, and indigo extract samples. The injection volume was 3 mg. Under a helium atmosphere, the temperature was increased from room temperature to 600℃ at a rate of 20℃ / min, held for 20 s, and then GC-MS was performed after complete pyrolysis. The thermal decomposition products were analyzed online by GC-MS using an HP-5MS column (30 mm × 0.25 µm × 0.25 mm). The column temperature was programmed: an initial temperature of 55℃ held for 1 min, followed by an increase to 250℃ at a rate of 15℃ / min and held for 3 min. The split ratio was 50:1; the flow rate was 1 mL / min; the carrier gas was helium; the injection port temperature was 300℃; and the transfer line temperature was 280℃. The mass spectrometer used an EI ion source; the ion source temperature was 250℃; the scanning range was 25–400 u. 4.6 Elemental Content Analysis The instrument used was a Thermo Fisher Scientific ICAP 7400 dual-channel inductively coupled plasma atomic emission spectrometer (ICP-AES), with an RF power of 1150 W, a cooling gas flow rate of 12.0 L / min, a nebulizer flow rate of 0.50 L / min, a nebulizer pressure of 210 kPa, and an auxiliary gas flow rate of 0.50 L / min. Radical axial observation mode was used. 0.2 g of sample powder was dissolved in 5 mL of nitric acid, and 1 mL of 30% hydrogen peroxide was added, followed by digestion and reconstitution to 50 mL. The standard curve was diluted with 2% UPS-grade nitric acid. The contents of As, Ca, Cd, Cu, Hg, and Pb in indigo paste powder, indigo paste acidified, and indigo extract samples were determined and analyzed. 4.7 UHPLC-TOF-MS Analysis Indigo extract powder, indigo extract acidified and organic components of indigo extract were analyzed by UHPLC-TOF-MS. Chromatographic conditions: UltiMate 3000 ultra-high pressure liquid chromatography; column (ACQUITY UPLC HSS T3 1.8 μm 2.1×100 mm); column temperature 40℃, sample loading 3 μL; mobile phase conditions: (1) positive ion mode: A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile; (2) negative ion mode: A: water (2 mM ammonium acetate); B: acetonitrile. Gradient elution: (0~2 min, 95% A; 2~30 min, 95%~7% A; 30~47 min, 7% A; 47~53 min, 7%~0% A; 53~54 min, 0~95% A; 54~58 min, 95% A). Mass spectrometry conditions: AB 5600 Triple TOF mass spectrometer; electrospray ionization source: positive and negative ion detection modes; primary acquisition range (m / z): 50-1200; bombardment energy: 30 eV, 10 secondary spectra every 50 ms. ESI ion source parameters were set as follows: nebulizer pressure (GS1): 60 Psi; auxiliary gas pressure: 60 Psi; curtain gas pressure: 35 Psi; temperature: 650℃; spray voltage: 5000 V (positive ion mode) or -4000 V (negative ion mode). 4.8 Analysis of the physicochemical properties of indigo extraction The indigo paste powder sample had an ash content of 49.53% and an acid-insoluble ash content of 10.58%. The significant difference between the two ash contents indicates the presence of large amounts of CaCO3, Ca(OH)2, and lime in the indigo paste powder. The high content of acid-insoluble ash suggests the presence of numerous non-lime impurities, such as SiO2. The indigo acid oxide sample had an ash content of 13.35% and an acid-insoluble ash content of 5.84%. This indicates that traditional indigo paste powder production, through hydrochloric acid treatment, can remove most of the lime impurities in the resulting indigo acid oxide product. The indigo extract sample produced by this patent had an ash content of only 1.6% and an acid-insoluble ash content of 1.35%, indicating that the main components of the high-purity indigo sample are organic. Thermogravimetric analysis of indigo paste powder, indigo acid oxide, indigo extract, and indigo standards showed that compared with indigo standards, the thermal stability of each indigo extract sample decreased, and the residual mass increased. The crude indigo extract had the largest residual mass, indicating a higher content of inorganic impurities. The residual mass of indigo acidified compounds was significantly lower than that of indigo paste powder, indicating that the acidification process can remove most of the inorganic impurities in indigo paste powder. The peak temperature of indigo extract in the range of 200-400℃ was lower than that of indigo standard, indicating that indigo extract contains organic compounds with lower boiling points. The residual mass was higher than that of indigo standard, indicating the presence of more high-boiling-point organic compounds. Py-GC-MS data analysis showed significant differences in the organic composition of the pyrolysis gas chromatography (280~600℃) analysis between traditional indigo processing and the indigo extract of this patent. Two compounds were identified in the indigo paste powder sample, 12 compounds were identified in the indigo acid compound, and 19 compounds were identified in the indigo extract sample. The small molecule compounds with fewer than 12 carbons mainly included: aldehydes (benzaldehyde, phenylacetaldehyde), alkenes (6,6-dimethylfuren, dextrorotatory terpenes); phenols (m-cresol), amides (N-acetanilide, naphthalene (1-methylnaphthalene), toluene (trimethylbenzene), indoles (indole, 7-methylindole, 2-indoleone, indigo); the compounds with 12~20 carbons mainly included alkenes (1-tetradecene, 1-nonadene) and aldehydes. Dodecaldehyde, naphthalenes (2,7-dimethylnaphthalene), indoles (indigo, tryptophanone), phenols (2,4-di-tert-butylphenol), acids (lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, 10-cis-heptadecenoic acid, transoleic acid, heptadecanic acid, linoleic acid, oleic acid, stearic acid, conjugated linoleic acid, linolenic acid), alkanes (hexadecane), alkynes (3-Dodecyne), tryptophanone, alcohols (phytol). Elemental analysis showed that indigo extract powder had the highest calcium content (275 g / kg), indigo acidified compound had a calcium content of 3 g / kg, and indigo extract had a calcium content of only 0.38 g / kg. The contents of As, Hg, and Pb were all below 10 mg / kg. The relative contents of each component were obtained by comparing with the NIST 08 mass spectrometry database and using the area normalization method. A total of 61 compounds were identified in the indigo acidified compound sample, and 185 compounds were identified in the indigo extract, mainly including nine categories of substances: acids, polysaccharides, flavonoids, alkaloids, glycosides, polyphenols, aldehydes, proteins, and esters. The results indicate that the indigo extract has significantly more active compounds than indigo extract powder and indigo acidified compound powder, suggesting that it has better antibacterial function. Example 5 Evaluation of the antibacterial function of Indigofera tinctoria extract 5.1 Materials: Aspergillus niger ( Aspergillus niger Staphylococcus aureus ( Staphyloco ccus aureus ), Escherichia coli ( Escherichia coli Sample 1 (Indigo extract powder, indigo content 3%), Sample 2 (Indigo extract powder acidified, indigo content 20%), Sample 3 (Indigo extract; indigo content 40%), Sample 4 (High purity indigo; indigo content 96.87%). 5.2 Main Reagents Potato glucose agar, LB nutrient agar, potato glucose solution, and LB broth were all purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd.; ammonium acetate, N,N-dimethylformamide, and dimethyl sulfoxide were all purchased from Sinopharm Chemical Reagent Co., Ltd.; methanol and acetonitrile were purchased from Aladdin Biochemical Reagent Co., Ltd., both of chromatographic purity. 5.3 Activation of microbial strains Staphylococcus aureus and Escherichia coli were inoculated into the bacterial activation solution in a clean bench and cultured at 37°C and 120 r / min for 24 h. The bacterial concentration was determined using a McFarland turbidimeter after a 10-fold dilution. The bacterial solution was then diluted with sterile physiological saline to a concentration of approximately 10. 6 ~10 8 CFU / mL, for later use. After activating the Aspergillus niger strain stored at -20℃ twice on PDA solid medium, spores were eluted from the slant with sterile physiological saline. The spore suspension concentration was determined using a McFarland turbidimeter, and the concentration was adjusted to 10. 6 ~10 8 CFU / mL, store at 4℃ for later use. 5.4 Determination of inhibition zones In a clean bench, 100 μL of Aspergillus niger spore suspension was spread onto a PDA solid agar plate, and 100 μL of Staphylococcus aureus and Escherichia coli suspensions were spread separately onto LB solid agar plates. Then, a sterilized Oxford cup (a round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm) was placed in the test plate and gently pressed to ensure complete contact with the petri dish. 20 mg / L natamycin was used as a positive control for Aspergillus niger inhibition, 0.1 mg / mL gentamicin as a positive control for Staphylococcus aureus and Escherichia coli inhibition, and DMSO as a negative control. Three replicates were performed for each sample and control. 100 μL of each of the following samples were transferred into Oxford cups: 2 mg / mL, 1 mg / mL, 0.5 mg / mL: Sample 1 (indigo extract powder, indigo content: 3.06%), Sample 2 (indigo extract powder acidified, indigo content: 20.13%), Sample 3 (indigo extract; indigo content: 40%), and Sample 4 (high-purity indigo sample; indigo content: 96.87%). After incubation at the optimal temperature for a period of time, the size of the inhibition zone was measured. 5.5 Determination of MIC and MBC The bacterial strain from the solid culture medium was inoculated into the liquid culture medium and incubated in an incubator for a period of time. The concentration of the liquid bacterial suspension was measured using a McFarland turbidimetric tube, and the concentration was adjusted to 1×10⁻⁶. 6CFU / mL. Using the micro-dose method in a 96-well plate, all sample solutions were prepared to a concentration of 5.12 mg / mL. 160 μL of sterile liquid culture medium and 40 μL of the prepared sample solution were added to the first well of each group. 100 μL of sterile liquid culture medium was added to wells 2-11, and 160 μL of sterile liquid culture medium and 40 μL of DMSO were added to well 12 as a blank control. Using a twofold dilution method, 100 μL of the liquid in well 1 was transferred to well 2, and so on, until well 11. 100 μL of the prepared liquid bacterial suspension was added to wells 1-11 respectively. At this point, the sample concentrations in wells 1-11 of the 96-well plate were 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL, respectively. The plates were incubated in a constant temperature incubator for 24 h to observe the results. The minimum inhibitory concentration (MIC) of the sample against the tested bacteria is the lowest concentration of the sample that does not become turbid on the plate. Based on the MIC test, 100 μL of each tube of culture medium from which no bacterial growth was observed was spread onto a solid culture medium and then placed in a constant temperature incubator for a period of time. The concentration of the sample corresponding to the culture medium from which no bacterial growth was observed after 24 h is the minimum bactericidal concentration (MBC) of this sample. 5.6 Determination of the half-maximal inhibitory concentration (IC50) The effect of different samples on the inhibition rate of indicator bacteria was determined by optical density method. Based on the MIC test, all sample solutions were prepared with a concentration of 5.12 mg / mL, and then diluted to obtain sample solutions with concentrations of 0.32, 0.64, 1.28, 1.60, 1.24, 2.56, 3.20, 3.84, 4.48, and 5.12 mg / mL. 20 μL of each of the above dilutions was transferred to a 96-well plate and mixed with 180 μL of a prepared liquid suspension of Staphylococcus aureus and Escherichia coli. At this point, the sample concentrations in the 96-well plates were 32, 64, 128, 160, 192, 224, 256, 320, 384, 448, and 512 μg / mL, respectively. The sample solutions were further diluted to obtain concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μg / mL. 20 μL of each dilution was transferred to a 96-well plate and mixed with 180 μL of prepared Aspergillus niger liquid suspension. The sample concentrations in the 96-well plates were then 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 μg / mL, respectively. The plates were incubated for 24 h, and the OD value of the bacterial suspension + sample was measured at 600 nm. A blank control was also performed, and the OD values ​​of the bacterial suspension + DMSO, OD LB + DMSO, and OD LB + sample were measured at 600 nm. The OD control represents the normal growth state of the indicator bacteria, while the actual OD value represents the growth of the indicator bacteria in the sample-containing medium. The IC50 value was determined by calculating the effect of the sample on the growth state of the indicator bacteria. The calculation formula is as follows: (1) In the formula: Actual OD value – The actual OD value of the sample at 600 nm OD bacterial suspension + sample — OD value at 600 nm of 180 μL bacterial suspension + 20 μL sample; OD LB+ sample — 180 μL liquid culture medium + 20 μL OD value of each sample at 600 nm. (2) In the formula: OD control – OD value of the reference sample at 600 nm OD bacterial suspension + DMSO — OD value at 600 nm for 180 μL bacterial suspension + 20 μL DMSO; ODLB+DMSO — OD value at 600 nm for 180 μL liquid culture medium + 20 μL DNSO. (3) In the formula: OD control – OD value of the reference sample at 600 nm Actual OD value – The actual OD value of the sample at 600 nm 5.7 Evaluation of the antibacterial activity of indigo extract All four samples showed good inhibitory effects against *Aspergillus niger* and *Staphylococcus aureus*, but weaker inhibitory effects against *Escherichia coli*, with the inhibitory effect decreasing as the sample concentration decreased. At a concentration of 2.0 mg / mL, the inhibition zone diameter against *Aspergillus niger* increased from 11.19 mm to 19.69 mm with increasing indigo content, the inhibition zone diameter against *Staphylococcus aureus* increased from 13.57 mm to 17.85 mm, and the inhibition zone diameter against *Escherichia coli* increased from 10.67 mm to 12.91 mm. The samples showed the best inhibitory effect against *Aspergillus niger*, with sample 1 having a minimum inhibitory concentration (MIC) of 2 μg / mL, while samples 2, 3, and 4 all had MICs of 1 μg / mL. Inhibition effects against *Staphylococcus aureus* and *Escherichia coli* were weak; samples 1 and 2 had MICs of 512 μg / mL against both bacteria, while samples 3 and 4 had MICs of 256 μg / mL against both bacteria. All samples showed significant bactericidal activity against Aspergillus niger. The lowest possible concentration (LC50) for sample 1 was 64 μg / mL, for sample 2 it was 32 μg / mL, for sample 3 it was 8 μg / mL, and for sample 4 it was 4 μg / mL. All samples exhibited the strongest inhibitory effect against Aspergillus niger, with IC50 values ​​ranging from 0.6 to 1.8 μg / mL. Samples 3 and 4 had the lowest IC50 values ​​of 0.6 μg / mL. The IC50 values ​​against Staphylococcus aureus ranged from 128 to 384 μg / mL, with sample 3 having the lowest IC50 value of 128 μg / mL. The IC50 values ​​against Escherichia coli ranged from 160 to 448 μg / mL, with sample 3 having the lowest IC50 value of 160 μg / mL. The antibacterial experiments showed that the inhibitory effect against Aspergillus niger, Staphylococcus aureus, and Escherichia coli increased with increasing indigo content. High-content natural indigo extract has a good inhibitory effect on Aspergillus niger, with an inhibition zone size of 19.69 mm, a MIC value of 1 μg / mL, and an MBC value of 4 μg / mL. It is expected to be developed and used as an antibacterial agent. Example 6 Evaluation of the antibacterial function of broccoli polyphenol extract The inhibitory activity of the broccoli polyphenol extract against *Escherichia coli*, *Staphylococcus aureus*, *Bacillus cereus*, and *Pseudomonas aeruginosa* was analyzed according to the method described in Example 5. The results are as follows: Figure 2As shown in the figure, at the same concentration, the inhibitory effects of the polyphenol extract on four bacteria were as follows: Staphylococcus aureus > Pseudomonas aeruginosa > Bacillus cereus > Escherichia coli. Among these bacteria, the polyphenol 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 polyphenol extract possesses good broad-spectrum antibacterial activity and will show great application potential in the fields of daily chemicals, pharmaceuticals, and feed additives. Example 7 Evaluation of the effect of polyphenol extract on wound healing function in mice Using mice as the research subjects, the promoting effect of broccoli polyphenol extract on wound healing was analyzed. The trends of body weight, wound healing area, and wound healing rate in different groups of mice are shown below. Figure 3 As shown. From Figure 3 As can be seen, the body weight of each group of mice increased over time, indicating that the polyphenol 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. By the end of the experiment, the wound healing rate of the polyphenol extract exceeded 98%. In conclusion, by analyzing wound healing indicators and mouse body weight, the excellent ability of polyphenol extract to promote wound healing has been clearly demonstrated, laying the foundation for its subsequent application in the fields of daily chemicals and pharmaceuticals.

Claims

1. A method for preparing multifunctional indigo and polyphenol extracts using the stems and leaves of *Indigofera tinctoria*, characterized in that... 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 10-80 cm on the same day, cut off 20-50 cm of woody stems with a hydraulic cutter, retain 10-30 cm of tender branches and leaves, and spray them evenly with water with pH 4-7 to remove dirt and other impurities. The second step is steam explosion hydrothermal extraction: the washed Stellera chamaejasminoides raw material is added to a steam explosion container, water with pH 4~7 is added, the volume ratio of Stellera chamaejasminoides raw material to water (g / mL) is 1:20~60, the steam pressure of the container reaches 0.5~2.5 MPa, the heat preservation time is 0.5~10 min, and then the pressure is released and the temperature is lowered to 20~50℃; The third step is solid-slag separation: the steam explosion hydrothermal extract is passed through a 10-40 mesh stainless steel screen, the filter residue is cleaned with tap water, and the filtrate is combined. Step 4, biological fermentation: Add 0.01~6% biological enzymes to the filtrate from step 3 for fermentation, with a fermentation pH of 5~10, a temperature of 30~60℃, and a time of 8~72 h; Step 5, membrane fractionation purification of indigo extract: The bio-fermentation broth from step 4 is clarified by passing it through a microfiltration membrane. The volume ratio of permeate to concentrate is 3~12:

1. The concentrate is separated by centrifugation, and the centrifuged precipitate is dried to obtain indigo extract with an indigo content greater than 40%, ash content less than 2%, calcium content less than 0.4 g / kg, lead (Pb) less than 10 mg / kg, and arsenic (As) less than 10 mg / kg. Step 6, Preparation of polyphenol extract: Combine the permeate from the microfiltration membrane in step 5 and the supernatant separated by centrifugation. Remove impurities by ultrafiltration membrane with a molecular weight of 10,000 to 50,000 Daltons. The volume ratio of permeate to concentrate is 3 to 10:

1. The permeate is then separated by nanofiltration membrane with a volume ratio of 3 to 6:

1. The concentrate is dried by microwave vacuum to obtain polyphenol extract with a total polyphenol content of more than 20%, of which chlorogenic acid is more than 5% and polysaccharides are more than 10%.

2. The method for preparing multifunctional indigo and polyphenol extracts using the stems and leaves of *Indigofera tinctoria* according to claim 1, characterized in that... The pH value mentioned in the first and second steps is adjusted to a water pH of 5-7 using any one of citric acid, acetic acid, oxalic acid, or sodium dihydrogen phosphate.

3. The method for preparing multifunctional indigo and polyphenol extracts from the stems and leaves of *Indigofera tinctoria* according to claim 1, characterized in that... The bioenzyme mentioned in step four is an enzyme capable of breaking the glycosidic bond in indole glycosides, including any one or any combination of the following: endogenous enzyme extract of strychnos nucifera leaf, Aspergillus niger enzyme, Acer truncatum cellulase and β-glucosidase.

4. The method for preparing multifunctional indigo and polyphenol extracts from the stems and leaves of *Indigofera tinctoria* according to claim 1, characterized in that... The microfiltration membrane mentioned in step four is any one of a ceramic membrane with a pore size of 50~200 nm, a nanoscale polymer organic membrane, or a stainless steel filter element with a pore size of 0.3~50 μm.

5. The endogenous enzyme extract from *Indigofera tinctoria* leaves according to claim 3, characterized in that, Fresh senna leaves were extracted by mechanical stirring with phosphate buffer solution (pH 5-7) and ice water at a solid-liquid ratio of 1:2-5, stirring speed of 3000-5000 r / min, and extraction time of 0.5-1 h. The pulp residue was then freeze-dried to obtain endogenous enzyme extracts from senna leaves.

6. The indigo extract according to claim 1, characterized by Py-GC-MS at 280~600℃, contains more than 15 organic compounds, and its UHPLC-Q-TOF-MS / MS characteristics identify nine classes of substances including acids, polysaccharides, flavonoids, alkaloids, glycosides, polyphenols, aldehydes, proteins, and esters, and is rich in more than 150 kinds of active organic substances; the indigo extract has good inhibitory function against Aspergillus niger, Staphylococcus aureus, and Escherichia coli, with a MIC value of 0.5~1.0 μg / mL and an MBC value of 2.0~4.0 μg / mL.

7. The indigo extract according to claim 1, characterized in that... Indigo extract possesses the natural dyeing function of indigo, is free of harmful aromatic amines, has a dyeing strength (for standard products) of 100 points, a K / S value greater than 20, and exhibits dyeing performance and fastness on cotton fabrics of dyeing depth grade 1, soaping fastness grade 3-4, perspiration fastness grade 3-5, dry rubbing fastness grade 3-5, and water fastness grade 3-5.

8. The polyphenol extract according to claim 1, characterized in that... Ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS / MS) was used to identify nine major phenolic substances, including at least one of three phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid), two phenylethanol glycosides (verbascoside, isoacteoside), and four flavonoids (isorhamnetin-3-O-glucoside, homoplantaginin, isorhamnetin, and hispidulin).

9. The polyphenol extract according to claim 1, characterized in that... The polyphenol extract has good antibacterial function against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus and Escherichia coli, with a MIC value of less than 45 μg / mL, and its wound healing ability in mice exceeds 98%.

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