Method for extracting flavones from neosinocalamus affinis leaves and application thereof

Abstract

The present application relates to the technical field of plant extract and the technical field of food processing, and particularly relates to a method for extracting flavones from bamboo leaves of Neosinocalamus affinis and application thereof. The method comprises the following steps: mixing bamboo leaf powder of Neosinocalamus affinis with pure water, and sequentially performing pulsed electric field treatment, ultrasonic treatment and enzymatic hydrolysis treatment; performing enzyme inactivation treatment after the enzymatic hydrolysis treatment; performing solid-liquid separation, collecting supernatant, and obtaining flavone extract from bamboo leaves of Neosinocalamus affinis; the bamboo leaf powder is two-year-old bamboo leaf powder of Neosinocalamus affinis. The method uses bamboo leaves of Neosinocalamus affinis as raw material, uses pure water as single extraction medium, performs pre-treatment on cell membrane by pulsed electric field to cause electroporation, further collapses cell wall and changes the conformation of cellulase by ultrasonic cavitation effect, and finally uses cellulase to specifically hydrolyze cellulose skeleton, so that the three time sequences are progressive and synergistic, and the flavone yield in the pure water system reaches 2.32-2.63%, which is comparable to organic solvent hot extraction and has no solvent residue.

Description

Technical Field

[0001] This invention relates to the fields of plant extract technology and food processing technology, and in particular to a method for extracting flavonoids from bamboo leaves and their application. Background Technology

[0002] Cizhu ( Bamboo Also known as Neosinocalamus affinis Bamboo (Phyllostachys edulis) is a distinctive clump-forming bamboo species widely distributed in southwestern my country (especially Yibin, Sichuan). The existing bamboo forest area is approximately 3.34 million mu (about 222,000 hectares). Cultivation and harvesting generate a large amount of bamboo leaf by-products, resulting in a vast resource reserve, but the comprehensive utilization rate is low. Bamboo leaves are rich in C-glycoside flavonoids such as vitexin, isovitexin, and gentiopicrin. Due to the synergistic effect of the phenolic hydroxyl structure and glycosidic bonds, they possess significant antioxidant and anti-inflammatory physiological activities. In 2014, bamboo leaf flavonoids were approved as a new food ingredient, possessing antioxidant, starch-inhibiting, and postprandial blood glucose-regulating activities, making them a high-quality natural raw material for developing functional foods.

[0003] Currently, organic solvent extraction is the most mainstream process for the industrial extraction of flavonoids from bamboo leaves. Ethanol solutions are typically used for hot reflux extraction. In addition, there are numerous reports of physical field-enhanced organic solvent extraction methods, such as ultrasound-assisted ethanol extraction and microwave-assisted ethanol extraction. These methods offer considerable flavonoid yields, but pose safety risks due to solvent residue, require additional desolvation equipment and processes, increasing production costs, and do not align with current trends towards clean labeling and natural, green consumption. In terms of physical field-enhanced extraction, technologies such as ultrasound-assisted extraction, microwave-assisted extraction, and high-voltage pulsed electric field-assisted extraction have been used to disrupt plant cell walls and shorten extraction time. However, existing research often combines physical field enhancement methods with organic solvents, failing to fundamentally solve the solvent residue problem, and the efficiency of pure aqueous phase physical field extraction is generally still unsatisfactory. For pure water extraction, using pure water as the solvent offers the highest safety and lowest cost. However, the main drawback of traditional water extraction is that the limited solubility of flavonoids in pure water and the significant barrier effect of the cellulose skeleton in plant cell walls lead to extremely low extraction efficiency and high impurity content in the extract, increasing the difficulty of subsequent purification.

[0004] In summary, existing technologies struggle to balance extraction safety and efficiency. While organic solvent methods offer high yields, the risk of solvent residue remains, making complete elimination difficult. Water extraction, though safe, suffers from low mass transfer efficiency, failing to meet the economic requirements of industrial production. Furthermore, research on systematic extraction processes specifically for the bamboo species *Bambusa textilis* is relatively scarce.

[0005] Therefore, developing a highly efficient and green extraction method for flavonoids from bamboo leaves that balances extraction safety and efficiency is of great practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical contradiction between the difficulty in simultaneously achieving organic solvent residue and low extraction efficiency of pure water in existing bamboo leaf flavonoid extraction methods. This invention provides a method and application for extracting flavonoids from *Bambusa textilis* leaves. The method uses *Bambusa textilis* leaves as raw material and pure water as the sole extraction medium. Pretreatment with a pulsed electric field (PEF) induces electroporation of the cell membrane, followed by ultrasonic cavitation to further disintegrate the cell wall and alter the conformation of cellulase. Finally, cellulase specifically hydrolyzes the cellulose skeleton. These three processes work sequentially and synergistically, achieving a flavonoid yield of 2.32-2.63% in the pure water system, comparable to organic solvent thermal extraction with no solvent residue. Based on this *Bambusa textilis* leaf extract, this invention also provides a sugar-controlling plant-based beverage. Through the synergistic effect of *Bambusa textilis* leaf flavonoids and mulberry leaf extract in inhibiting α-glucosidase, combined with bitter melon extract to assist in improving sugar metabolism, a dual-pathway sugar control approach is achieved, inhibiting sugar decomposition and improving sugar metabolism. Simultaneously, a complex sweetener of erythritol and steviol glycosides masks the bitterness, solving the taste problem of beverages with high flavonoid content. In addition, the bamboo leaf extract of this *Cizhu* can also be added to foods such as jelly and yogurt as a food additive, giving the products both antioxidant and sugar-controlling functions, thus realizing the high-value utilization of *Cizhu* processing residues.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for extracting flavonoids from bamboo leaves of *Bambusa textilis*, the extraction method comprising the following steps: mixing *Bambusa textilis* bamboo leaf powder with pure water, and sequentially subjecting the mixture to pulsed electric field treatment, ultrasonic treatment, and enzymatic hydrolysis; after the enzymatic hydrolysis, performing enzyme inactivation treatment; separating the solid and liquid components, collecting the supernatant, and obtaining a flavonoid extract from *Bambusa textilis* bamboo leaves; wherein the *Bambusa textilis* bamboo leaf powder is powder from two-year-old *Bambusa textilis* bamboo leaves.

[0008] Preferably, the ratio of the bamboo leaf powder to pure water is 1g:10~30mL.

[0009] Preferably, the conditions for pulsed electric field processing are: electric field strength 10~50kV / cm, pulse width 10~50μs, pulse frequency 50~300Hz, and effective processing time 50~300μs.

[0010] More preferably, the conditions for pulsed electric field processing are: electric field strength 20~35kV / cm, pulse width 20~30μs, and pulse frequency 100~200Hz.

[0011] More preferably, the pulsed electric field treatment is followed by a resting period of 5-15 minutes before the ultrasonic treatment is performed.

[0012] Preferably, the conditions for the ultrasonic treatment are: temperature of 15~30℃, ultrasonic power of 300~500W, frequency of 40kHz, and ultrasonic time of 10~30min.

[0013] More preferably, the conditions for the ultrasonic treatment are: ultrasonic power 350~450W, ultrasonic time 15~20min.

[0014] Preferably, the enzymatic hydrolysis treatment is as follows: after ultrasonic treatment, the pH is adjusted to 4.5~5.5 using a pH adjuster, cellulase is added, and enzymatic hydrolysis is carried out at a temperature of 45~55℃ for 45~90 minutes.

[0015] Preferably, the pH adjuster is selected from one or more of citric acid, malic acid, lactic acid, and tartaric acid.

[0016] Preferably, the amount of cellulase added is 0.5-2.5% of the mass of the bamboo leaf powder, and the enzyme activity is ≥400U / mg.

[0017] Preferably, the solid-liquid separation is centrifugal separation or vacuum filtration.

[0018] Preferably, the extraction method comprises an extraction cycle consisting of ultrasonic treatment, enzymatic hydrolysis, enzyme inactivation, and solid-liquid separation.

[0019] Preferably, the extraction cycle is repeated 2 to 3 times, and the ratio of filter residue to pure water in each extraction cycle is 1g:10~30mL. The total time for ultrasonic treatment and enzymatic hydrolysis is 60~110min.

[0020] Preferably, the extraction method further includes concentrating the extract of flavonoids from bamboo leaves under reduced pressure at a temperature of 50-55°C and a pressure of -0.10-0.08 MPa to 0.2-0.5 times the original volume to obtain a concentrated extract of flavonoids from bamboo leaves.

[0021] Preferably, the concentrated flavonoid solution of *Bambusa textilis* leaves is spray-dried to obtain *Bambusa textilis* leaf flavonoid extract powder.

[0022] Preferably, the spray drying conditions include: an inlet air temperature of 160~180℃, an outlet air temperature of 80~90℃, and a feed rate of 40~60 mL / min.

[0023] Secondly, the present invention provides a flavonoid extract of bamboo leaves prepared by the extraction method described in the present invention.

[0024] Thirdly, the present invention provides a plant-based beverage composition comprising: a flavonoid extract of bamboo leaves, a mulberry leaf extract, a bitter melon extract, a licorice extract, a sweetener, an acidulant, a stabilizer, and water; The flavonoid extract of bamboo leaves mentioned herein is the same as the flavonoid extract of bamboo leaves mentioned in this invention.

[0025] Preferably, the sweetener is a mixture of erythritol and steviol glycosides, wherein the mass ratio of erythritol to steviol glycosides is 10-20:1.

[0026] Preferably, the acidulant is selected from one or more of citric acid, malic acid, lactic acid, and tartaric acid.

[0027] Preferably, the stabilizer is selected from one or more of xanthan gum, sodium carboxymethyl cellulose, sodium alginate, and carrageenan.

[0028] Preferably, the pH of the plant-based beverage composition is 3.5 to 5.0, and more preferably 4.0 to 4.2.

[0029] Preferably, per 1000 mL of beverage, the mass-volume percentage of each component in the plant-based beverage composition is as follows: 0.1-2.0% of *Bambusa textilis* leaf flavonoid extract, 0.2-1.5% of mulberry leaf extract, 0.1-1.0% of bitter melon extract, 0.1-0.5% of licorice extract, 3.0-8.0% of erythritol, 0.01-0.05% of steviol glycosides, 0.1-0.3% of citric acid, 0.01-0.05% of xanthan gum, with the remainder being purified water.

[0030] More preferably, the mass-volume percentage of each component in the plant-based beverage composition per 1000 mL of beverage is as follows: 1.0% of bamboo leaf flavonoid extract, 0.5% of mulberry leaf extract, 0.3% of bitter melon extract, 0.2% of licorice extract, 4.0% of erythritol, 0.02% of steviol glycosides, 0.2% of citric acid, 0.02% of xanthan gum, and the balance being purified water.

[0031] Fourthly, the present invention provides the application of the flavonoid extract of bamboo leaves as described in the present invention or the plant-based beverage composition described in the present invention in the preparation of food, wherein the flavonoid extract of bamboo leaves as a food additive is added to the food in the form of a concentrated liquid or powder, and the amount added is 0.1 to 1.0% of the total mass of the food.

[0032] Preferably, the food is selected from one or more of jelly, yogurt, nutrition bars, biscuits, or functional candies.

[0033] In the above technical solution, the extraction method of bamboo leaves flavonoids of the present invention uses two-year-old bamboo leaves of *Phyllostachys edulis* as raw material, and optimizes the basic activity of the raw material by limiting the bamboo species and growth years. The cultivated bamboo leaves of *Phyllostachys edulis* are waste byproducts, making the raw material readily available and inexpensive. Two-year-old bamboo leaves have the highest content of C-glycoside flavonoids such as vitexin and gentianin. Compared with common bamboo species such as *Phyllostachys pubescens* and *Phyllostachys nigra*, the raw material has stronger inherent antioxidant and sugar-controlling activities. Using this as a raw material not only ensures extraction efficiency from the source, but also transforms the bamboo leaf processing residues generated from cultivation and thinning into high-value-added products, realizing the high-value utilization of this unique bamboo species resource.

[0034] Secondly, the ternary coupled pure water preparation method of this invention achieves efficient and green extraction through a segmented synergistic cell wall disruption mechanism of pulsed electric field-ultrasound-cellulase. The pulsed electric field induces reversible electroporation of the cell membrane within microseconds, preferentially breaking down the membrane barrier for intracellular flavonoid release; subsequently, the microjets and shock waves generated by ultrasonic cavitation further disintegrate the cell wall skeleton, while simultaneously inducing conformational changes in cellulase, increasing the enzyme's affinity for the substrate and its binding sites; finally, cellulase specifically hydrolyzes the cellulose skeleton, causing complete cell wall disintegration. These three processes work synergistically according to a progressive logic of creating channels, expanding fissures, and directional degradation, forming a complete physical-biological mass transfer chain. Furthermore, no organic solvents are used throughout the process, eliminating the risk of solvent residue. The flavonoid yield is 2.32~2.63%, which is comparable to the extraction efficiency of the traditional 70% ethanol extraction method. Moreover, the low-temperature process effectively protects the phenolic hydroxyl structure of the heat-sensitive C-glycoside flavonoids, significantly improving the DPPH and ABTS free radical scavenging ability and the α-amylase and α-glucosidase inhibitory activity of the bamboo leaf extract of this invention, thus taking into account the triple advantages of safety, high efficiency and activity retention.

[0035] Furthermore, the bamboo leaf extract of this invention can be specifically applied to two major scenarios: plant-based beverages and food additives. In beverages, the C-glycoside structure of bamboo leaf flavonoids and DNJ from mulberry leaf extract synergistically enhance α-glucosidase inhibition. Combined with bitter melon extract to improve insulin sensitivity, a dual-pathway sugar control system that inhibits carbohydrate breakdown and improves glucose metabolism is constructed. Simultaneously, erythritol and steviol glycosides neutralize the bitterness of the raw materials, xanthan gum inhibits storage stratification, and the flavonoid retention rate exceeds 95% after 30 days of storage at 4°C. In addition, the bamboo leaf extract of this invention can be formulated into concentrated liquid or powder form as an additive, suitable for various foods such as jelly, yogurt, and nutrition bars, expanding the application scenarios of bamboo leaf flavonoids and building a complete high-value industrial chain for bamboo resources.

[0036] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0037] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the process flow for the green extraction method of flavonoids from bamboo leaves of the present invention. Detailed Implementation

[0038] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0039] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] In a first aspect, the present invention provides a method for extracting flavonoids from bamboo leaves of *Bambusa textilis*, the extraction method comprising the following steps: mixing bamboo leaf powder of *Bambusa textilis* with pure water, and sequentially subjecting it to pulsed electric field treatment, ultrasonic treatment and enzymatic hydrolysis treatment; after the enzymatic hydrolysis treatment, performing enzyme inactivation treatment; solid-liquid separation, collecting the supernatant to obtain flavonoid extract from bamboo leaves of *Bambusa textilis*; wherein the bamboo leaf powder of *Bambusa textilis* is powder from two-year-old bamboo leaves of *Bambusa textilis*.

[0043] The ternary coupled pure water preparation method of this invention achieves efficient and green extraction through a segmented synergistic cell wall disruption mechanism of pulsed electric field-ultrasound-cellulase. The pulsed electric field induces reversible electroporation of the cell membrane within microseconds, preferentially breaking down the membrane barrier for intracellular flavonoid release. Subsequently, the microjets and shock waves generated by ultrasonic cavitation further disintegrate the cell wall skeleton, while simultaneously inducing conformational changes in cellulase, increasing the enzyme's affinity for the substrate and its binding sites. Finally, cellulase specifically hydrolyzes the cellulose skeleton, causing complete cell wall disintegration. These three processes work synergistically according to a progressive logic of channel creation, crack expansion, and directional degradation, forming a complete physical-biological mass transfer chain. Furthermore, no organic solvents are used throughout the process, eliminating the risk of solvent residue. The flavonoid yield is 2.32~2.63%, which is comparable to the extraction efficiency of the traditional 70% ethanol extraction method. Moreover, the low-temperature process effectively protects the phenolic hydroxyl structure of the heat-sensitive C-glycoside flavonoids, significantly improving the DPPH and ABTS free radical scavenging ability and the α-amylase and α-glucosidase inhibitory activity of the bamboo leaf extract of this invention, thus taking into account the triple advantages of safety, high efficiency and activity retention.

[0044] The ratio of bamboo leaf powder to pure water in this invention is 1g:10~30mL, which ensures the full dissolution of flavonoids while controlling production costs.

[0045] The pulsed electric field treatment conditions described in this invention are: electric field strength 10~50 kV / cm, pulse width 10~50 μs, pulse frequency 50~300 Hz, and effective treatment time 50~300 μs. Under these conditions, the pulsed electric field applies a high-voltage electric pulse to the cell membrane within microseconds, causing reversible or irreversible electroporation of the cell membrane. This instantaneously disrupts the cell membrane's barrier structure, establishing a preferential channel for the release of intracellular flavonoids.

[0046] In a preferred embodiment of the present invention, the conditions for pulsed electric field processing are: electric field strength 20~35kV / cm, pulse width 20~30μs, and pulse frequency 100~200Hz.

[0047] The effective processing time described in this invention is the product of the total number of pulses and the pulse width.

[0048] The temperature rise during PEF treatment is typically ≤5℃, requiring no additional cooling. After pulsed electric field treatment as described in this invention, the cells are allowed to stand for 5~15 minutes before undergoing ultrasonic treatment, which enables the cell contents to be fully dissolved, providing a better material basis for subsequent ultrasonic and enzymatic hydrolysis processes.

[0049] The cavitation effect of ultrasound generates microjets and shock waves that can further break down the cell wall structure weakened by electroporation. The ultrasound treatment conditions described in this invention are: temperature 15~30℃, ultrasound power 300~500W, frequency 40kHz, and ultrasound time 10~30min, thereby further breaking down the cell wall structure and fully exposing the cellulase binding sites. At the same time, ultrasound treatment can induce conformational changes in cellulase, increasing the affinity and binding sites between the enzyme and the substrate.

[0050] In a preferred embodiment of the present invention, the conditions for ultrasonic treatment are: ultrasonic power 350~450W, ultrasonic time 15~20min.

[0051] The enzymatic hydrolysis process described in this invention is as follows: after ultrasonic treatment, the pH is adjusted to 4.5-5.5 using a pH adjuster, cellulase is added, and enzymatic hydrolysis is carried out at a temperature of 45-55℃ for 45-90 minutes. The cellulase specifically hydrolyzes the cellulose skeleton in the cell wall, causing the cell wall structure to completely disintegrate and the flavonoids to fully dissolve.

[0052] The pH adjuster described in this invention is selected from one or more of citric acid, malic acid, lactic acid, and tartaric acid.

[0053] The amount of cellulase added in this invention is 0.5-2.5% of the mass of the bamboo leaf powder, and the enzyme activity is ≥400U / mg.

[0054] The enzyme inactivation treatment conditions in this invention include: after the enzymatic hydrolysis treatment is completed, the liquid is heated to 90°C and maintained for 10-15 minutes.

[0055] The solid-liquid separation described in this invention is centrifugal separation or vacuum filtration.

[0056] To ensure sufficient dissolution of flavonoids, the extraction method of this invention comprises an extraction cycle consisting of ultrasonic treatment, enzymatic hydrolysis, enzyme inactivation, and solid-liquid separation. Preferably, the extraction cycle is repeated 2-3 times, with the material-to-liquid ratio of the filter residue to pure water being 1g:10-30mL in each cycle. The total time for ultrasonic treatment and enzymatic hydrolysis is 60-110min. The filtrates obtained from each solid-liquid separation are combined to obtain the flavonoid extract from *Bambusa textilis* leaves. Maintaining a consistent material-to-liquid ratio in each extraction cycle ensures that the mass transfer driving force of flavonoids is similar in each extraction, thereby guaranteeing the stability and repeatability of the total extraction rate.

[0057] The extraction method of the present invention further includes concentrating the extract of flavonoids from bamboo leaves under reduced pressure at a temperature of 50~55℃ and a pressure of -0.10~-0.08MPa to 0.2~0.5 times the original volume to obtain a concentrated extract of flavonoids from bamboo leaves.

[0058] The present invention involves spray drying the concentrated flavonoid solution of bamboo leaves of *Bambusa textilis* to obtain a powder of flavonoid extract from bamboo leaves of *Bambusa textilis*.

[0059] The spray drying conditions described in this invention include: an inlet air temperature of 160~180℃, an outlet air temperature of 80~90℃, and a feed rate of 40~60 mL / min.

[0060] Secondly, the present invention provides a flavonoid extract of bamboo leaves prepared by the extraction method described in the present invention.

[0061] Thirdly, the present invention provides a plant-based beverage composition comprising: a flavonoid extract of bamboo leaves, a mulberry leaf extract, a bitter melon extract, a licorice extract, a sweetener, an acidulant, a stabilizer, and water; The flavonoid extract of bamboo leaves mentioned herein is the same as the flavonoid extract of bamboo leaves mentioned in this invention.

[0062] The sweetener of the present invention is a mixture of erythritol and steviol glycosides, wherein the mass ratio of erythritol to steviol glycosides is 10~20:1.

[0063] The acidulant described in this invention is selected from one or more of citric acid, malic acid, lactic acid, and tartaric acid.

[0064] The stabilizer described in this invention is selected from one or more of xanthan gum, sodium carboxymethyl cellulose, sodium alginate, and carrageenan.

[0065] The pH of the plant-based beverage composition of the present invention is 3.5 to 5.0, preferably 4.0 to 4.2.

[0066] Based on per 1000mL of beverage, the mass-volume percentage of each component in the plant-based beverage composition of this invention is as follows: 0.1-2.0% of *Bambusa textilis* leaf flavonoid extract, 0.2-1.5% of mulberry leaf extract, 0.1-1.0% of bitter melon extract, 0.1-0.5% of licorice extract, 3.0-8.0% of erythritol, 0.01-0.05% of steviol glycosides, 0.1-0.3% of citric acid, 0.01-0.05% of xanthan gum, with the remainder being purified water.

[0067] Based on per 1000mL of beverage, the mass-volume percentage of each component in the plant-based beverage composition of this invention is as follows: 1.0% of bamboo leaf flavonoid extract, 0.5% of mulberry leaf extract, 0.3% of bitter melon extract, 0.2% of licorice extract, 4.0% of erythritol, 0.02% of steviol glycosides, 0.2% of citric acid, 0.02% of xanthan gum, and the balance being purified water.

[0068] Fourthly, the present invention provides the application of the flavonoid extract of bamboo leaves as described in the present invention or the plant-based beverage composition described in the present invention in the preparation of food, wherein the flavonoid extract of bamboo leaves as a food additive is added to the food in the form of a concentrated liquid or powder, and the amount added is 0.1 to 1.0% of the total mass of the food.

[0069] This invention relates to the targeted application of *Bambusa textilis* leaf extract in two main scenarios: plant-based beverages and food additives. In beverages, the C-glycoside structure of *Bambusa textilis* leaf flavonoids synergistically enhances the inhibition of α-glucosidase with DNJ extracted from mulberry leaves. Combined with bitter melon extract to improve insulin sensitivity, a dual-pathway sugar control system is constructed, inhibiting carbohydrate breakdown and improving glucose metabolism. Simultaneously, erythritol and steviol glycosides neutralize the bitterness of the raw materials, xanthan gum inhibits storage stratification, and the flavonoid retention rate exceeds 95% after 30 days of storage at 4°C. Furthermore, the *Bambusa textilis* leaf extract of this invention can be formulated into concentrated liquid or powder form as an additive, suitable for various foods such as jellies, yogurts, and nutrition bars, expanding the application scenarios of bamboo leaf flavonoids and constructing a complete high-value industrial chain for bamboo resources.

[0070] The food products described in this invention are selected from one or more of the following: jelly, yogurt, nutrition bars, biscuits, or functional candies.

[0071] In this invention, the room temperature is 15~30℃.

[0072] The present invention will be described in detail below through examples, but the scope of protection of the present invention is not limited thereto. In the following examples, the drugs and pharmaceuticals are all conventional commercially available products.

[0073] • Raw material preparation: Collect healthy two-year-old bamboo leaves (from bamboo base in Changning County, Yibin City, Sichuan Province), remove impurities, dry in a constant temperature drying oven at 37℃ for 24 hours to reduce the moisture content to <10%, pulverize using a high-speed universal pulverizer, and pass through an 80-mesh sieve to obtain two-year-old bamboo leaf powder.

[0074] The mulberry leaf extract, bitter melon extract, and licorice extract used in this invention have no special raw material source or preparation process limitations. They can all be prepared by conventional water extraction, alcohol extraction, or ultrasound-assisted extraction processes disclosed in the prior art. Any commercially available food-grade / health product-grade mulberry leaf, bitter melon, and licorice extract can be used for compounding. Those skilled in the art can freely choose the corresponding commercially available products according to actual production needs, without limiting the brand, supplier, or refined preparation steps of the extract.

[0075] • Used in Application Example 1 of this invention: Mulberry leaf extract: Brand: Snowt Biotechnology, Specification: 10:1, Fufeng Snowt Biotechnology Co., Ltd.; Bitter melon extract: Brand: SENTIAN, food grade, Shaanxi SENTIAN Biotechnology Co., Ltd.; Licorice extract: Brand: Snowt Biotechnology, Specification: 10:1, Fufeng Snowt Biotechnology Co., Ltd.

[0076] Example 1 S1 Pulsed Electric Field Treatment: Weigh 1000g of two-year-old bamboo leaf powder and add it to 20L of deionized water at a material-to-liquid ratio of 1g:20mL. Stir well to obtain a material solution. Place the material solution in a pulsed electric field treatment chamber, setting the electric field strength to 25kV / cm, the pulse width to 25μs, the pulse frequency to 150Hz, and the effective treatment time to 150μs. After treatment, let it stand for 10min to allow the material solution temperature to return to room temperature (15~30℃).

[0077] S2 Ultrasonic treatment: The liquid material treated with pulsed electric field in step S1 is placed in an ultrasonic extraction tank and ultrasonically treated for 15 minutes at a temperature of 25℃, a frequency of 40kHz, and a power of 400W.

[0078] S3 Enzymatic hydrolysis treatment: After ultrasonic treatment, the pH of the solution was adjusted to 5.0 with 50% citric acid solution. 15g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.015:1) was added. The solution was hydrolyzed at a constant temperature of 50℃ for 60min. The solution was stirred every 10min during the hydrolysis process.

[0079] S4 Solid-Liquid Separation and Concentration: After enzymatic hydrolysis, the liquid is heated to 90℃ and kept at 10 min to inactivate the enzyme; a horizontal spiral centrifuge is used to centrifuge at 4000 rpm for 10 min, and the supernatant is collected. Add 10L of deionized water to the filter residue and repeat step S2 ultrasonic treatment and step S3 enzymatic hydrolysis treatment once each. After enzymatic hydrolysis, heat the liquid to 90℃ and keep it for 10min to inactivate the enzyme. Centrifuge at 4000rpm for 10min using a horizontal spiral centrifuge and collect the supernatant. Combine the two supernatants. The combined supernatant was concentrated under reduced pressure at a temperature of 55℃ and a pressure of -0.09MPa until the volume of the concentrate was 2L.

[0080] S5 Drying: The concentrate from step S4 is spray-dried under the conditions of inlet air temperature of 170℃, outlet air temperature of 85℃, and feed rate of 50mL / min to obtain 25.8g of dried powder, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B1.

[0081] Example 2 The procedure was carried out in accordance with Example 1, except that "add 20L of deionized water at a material-to-liquid ratio of 1:20" in step S1 was replaced with "add 10L of deionized water at a material-to-liquid ratio of 1:10". Other conditions remained unchanged, and 23.2g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B2.

[0082] Example 3 The procedure was carried out in accordance with Example 1, except that "adding 20L of deionized water at a material-to-liquid ratio of 1:20" in step S1 was replaced with "adding 30L of deionized water at a material-to-liquid ratio of 1:30", while other conditions remained unchanged. 24.1g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B3.

[0083] Example 4 The procedure was carried out in accordance with Example 1, except that in step S1, "setting the electric field strength to 25kV / cm, the pulse width to 25μs, the pulse frequency to 150Hz, and the effective processing time to 150μs" was replaced with "setting the electric field strength to 10kV / cm, the pulse width to 10μs, the pulse frequency to 50Hz, and the effective processing time to 50μs". Other conditions remained unchanged, and 23.8g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B4.

[0084] Example 5 The procedure was carried out in accordance with Example 1, except that in step S1, "setting the electric field strength to 25 kV / cm, the pulse width to 25 μs, the pulse frequency to 150 Hz, and the effective processing time to 150 μs" was replaced with "setting the electric field strength to 50 kV / cm, the pulse width to 50 μs, the pulse frequency to 300 Hz, and the effective processing time to 300 μs". Other conditions remained unchanged, and 26.3 g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B5.

[0085] Example 6 The procedure was carried out in accordance with Example 1, except that in step S2, "ultrasonic treatment for 15 minutes at a temperature of 25°C, a frequency of 40kHz, and a power of 400W" was replaced with "ultrasonic treatment for 10 minutes at a temperature of 15°C, a frequency of 40kHz, and a power of 300W". Other conditions remained unchanged, and 24.5g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B6.

[0086] Example 7 The procedure was carried out in accordance with Example 1, except that the phrase "ultrasonic treatment for 15 minutes at a temperature of 25°C, a frequency of 40kHz, and a power of 400W" in step S2 was replaced with "ultrasonic treatment for 30 minutes at a temperature of 30°C, a frequency of 40kHz, and a power of 500W". All other conditions remained the same, and 25.5g of dried powder was obtained, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as B7.

[0087] Example 8 The procedure was carried out according to Example 1, except that in step S3, "adjusting the pH of the solution to 5.0 with a 50% citric acid solution, adding 15g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.015:1), and enzymatically hydrolyzing at 50°C for 60min" was replaced with "adjusting the pH of the solution to 4.5 with a 50% citric acid solution, adding 5g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.005:1), and enzymatically hydrolyzing at 45°C for 45min". Other conditions remained unchanged, and 23.5g of dried powder was obtained, which is the flavonoid extract of *Bambusa textilis* leaves (food additive grade), denoted as B8.

[0088] Example 9 The procedure was carried out according to Example 1, except that in step S3, "adjusting the pH of the solution to 5.0 with a 50% citric acid solution, adding 15g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.015:1), and enzymatically hydrolyzing at 50°C for 60min" was replaced with "adjusting the pH of the solution to 5.5 with a 50% citric acid solution, adding 25g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.025:1), and enzymatically hydrolyzing at 55°C for 90min". Other conditions remained unchanged, and 26.2g of dried powder was obtained, which is the flavonoid extract of *Bambusa textilis* leaves (food additive grade), denoted as B9.

[0089] Comparative Example 1 Traditional ethanol extraction method: Take 100g of *Bambusa textilis* leaf powder, add 2000mL of 70% ethanol aqueous solution, and extract by hot reflux at 70℃ for 2h. Filter, collect the filtrate, and extract the residue again under the same conditions. Combine the two filtrates and concentrate under reduced pressure at 60℃ and -0.09MPa to recover ethanol, obtaining a concentrated solution. Spray dry the concentrated solution at an inlet air temperature of 170℃, an outlet air temperature of 85℃, and a feed rate of 50mL / min to obtain 24.0g of dried powder, which is the *Bambusa textilis* leaf flavonoid extract, denoted as D1.

[0090] Comparative Example 2 Pure water extraction method: Take 100g of two-year-old bamboo leaf powder, add 2000mL of deionized water, and stir and extract for 2h at 60℃ and 200rpm. Filter and collect the filtrate. Extract the residue again under the same conditions. Combine the two filtrates and concentrate under reduced pressure at 55℃ and -0.09MPa to a volume of 200mL. Spray dry the concentrate at an inlet air temperature of 170℃, an outlet air temperature of 85℃, and a feed rate of 50mL / min to obtain 15.2g of dried powder, which is the bamboo leaf flavonoid extract, denoted as D2.

[0091] Comparative Example 3 Weigh 100g of powdered two-year-old bamboo leaves and add 2000mL of deionized water at a material-to-liquid ratio of 1:20. Stir well to obtain a liquid, place it in an ultrasonic extraction tank, and ultrasonically treat it for 60min at a temperature of 60℃, a frequency of 40kHz, and a power of 400W. Centrifuge it for 10min at a speed of 4000rpm using a horizontal spiral centrifuge, collect the supernatant, add 1000mL of deionized water to the filter residue and repeat the extraction once. Combine the two supernatants. Concentrate the combined supernatant under reduced pressure at a temperature of 55℃ and a pressure of -0.09MPa to a concentrated liquid volume of 200mL.

[0092] The concentrate was spray-dried at an inlet air temperature of 170℃, an outlet air temperature of 85℃, and a feed rate of 50mL / min to obtain 18.5g of dried powder, which is the flavonoid extract of bamboo leaves from *Cizhu*, denoted as D3.

[0093] Comparative Example 4 Weigh 100g of powdered two-year-old bamboo leaves, add 2000mL of deionized water at a material-to-liquid ratio of 1:20, stir well to obtain a material solution, adjust the pH of the material solution to 5.0 with citric acid, add 1g of cellulase with an enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.01:1), and enzymatically hydrolyze at a constant temperature of 50℃ for 60min, stirring the material solution once every 10min during the enzymatic hydrolysis process.

[0094] After enzymatic hydrolysis, the solution was heated to 90℃ and held for 10 minutes to inactivate the enzyme. The solution was centrifuged at 4000 rpm for 10 minutes using a horizontal spiral centrifuge. The supernatant was collected, and the filter residue was extracted once more with 1000 mL of deionized water. The two supernatants were combined. The combined supernatant was concentrated under reduced pressure at 55℃ and -0.09 MPa to a volume of 200 mL.

[0095] The concentrate was spray-dried at an inlet air temperature of 170℃, an outlet air temperature of 85℃, and a feed rate of 50mL / min to obtain 19.2g of dried powder, which is the flavonoid extract of bamboo leaves from *Cizhu*, denoted as D4.

[0096] Comparative Example 5 The procedure was carried out in accordance with Example 1, except that the pulsed electric field treatment in step S1 was not performed. Instead, the powder of two-year-old bamboo leaves was directly subjected to ultrasonic treatment, enzymatic hydrolysis, solid-liquid separation and concentration and drying, with other conditions remaining unchanged, to obtain 23.0g of dried powder, which is the bamboo leaf flavonoid extract (food additive grade), denoted as D5.

[0097] Comparative Example 6 S1 Ultrasonic Treatment: Weigh 1000g of two-year-old bamboo leaf powder, add 20L of deionized water at a material-to-liquid ratio of 1:20, stir evenly to obtain a material solution, place the material solution in an ultrasonic extraction tank, and ultrasonically treat for 15min at a temperature of 25℃, a frequency of 40kHz, and a power of 400W.

[0098] S2 Pulsed Electric Field Treatment: The ultrasonically treated liquid from step S1 is placed in a pulsed electric field treatment chamber. The electric field strength is set to 25 kV / cm, the pulse width to 25 μs, the pulse frequency to 150 Hz, and the effective treatment time to 150 μs. After treatment, the liquid is allowed to stand for 10 minutes to allow the temperature to return to room temperature (15~30℃).

[0099] S3 enzymatic hydrolysis treatment: After pulsed electric field treatment, the pH of the solution was adjusted to 5.0 with 50% citric acid solution. 15g of cellulase with enzyme activity ≥400U / mg (enzyme to substrate mass ratio of 0.015:1) was added. The solution was hydrolyzed at a constant temperature of 50℃ for 60min. The solution was stirred once every 10min during the hydrolysis process.

[0100] S4 Solid-Liquid Separation and Concentration: After enzymatic hydrolysis, the liquid was heated to 90℃ and held for 10 minutes to inactivate the enzyme; a horizontal spiral centrifuge was used to centrifuge at 4000 rpm for 10 minutes, and the supernatant was collected. The filter residue was added to 10L of deionized water for repeated extraction, and the two supernatants were combined; the combined supernatant was concentrated under reduced pressure at 55℃ and -0.09MPa to a volume of 2L.

[0101] S5 Drying: The concentrate from step S4 is spray-dried under the conditions of inlet air temperature of 170℃, outlet air temperature of 85℃, and feed rate of 50mL / min to obtain 23.3g of dried powder, which is the flavonoid extract of bamboo leaves (food additive grade), denoted as D6.

[0102] Comparative Example 7 The procedure was carried out in accordance with Example 1, except that the raw material "two-year-old bamboo leaf powder" was replaced with "one-year-old bamboo leaf powder", while other processing conditions remained unchanged, resulting in 21.8g of dried powder, which is the bamboo leaf flavonoid extract (food additive grade), denoted as D7.

[0103] Detection Example 1 The total flavonoid content and flavonoid yield of the bamboo leaf flavonoid extracts obtained in Examples 1-9 and Comparative Examples 1-7 were detected using the following methods: • Determination of total flavonoid content: The total flavonoid content was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method.

[0104] • Yield (%) = [Mass of Bamboo Leaf Flavonoid Extract (g) / Mass of Bamboo Leaf Powder (g)] × 100%; The results are shown in Table 1.

[0105] Table 1 As shown in Table 1, the total flavonoid content and yield of the *Bambusa textilis* leaf flavonoid extracts obtained in Examples 1-9 of this invention are significantly superior to the conventional water extraction methods used in Comparative Examples 2-4. Specifically, the *Bambusa textilis* leaf flavonoid extract obtained in Example 1 has a total flavonoid content of 24.5 mg RE / g and a yield of 2.58%, which is far higher than the total flavonoid content and yield of the *Bambusa textilis* leaf flavonoid extract obtained in Comparative Example 2 using pure water extraction. It is also significantly superior to the extraction effects of Comparative Example 3 using only ultrasound and Comparative Example 4 using only enzymatic hydrolysis.

[0106] The total flavonoid content of the *Bambusa textilis* leaf flavonoid extract prepared in Comparative Example 5 without pulsed electric field pretreatment was 22.8 mg RE / g, with a yield of 2.30%. The total flavonoid content of the *Bambusa textilis* leaf flavonoid extract prepared in Comparative Example 6, with the pulsed electric field pretreatment and ultrasonic treatment sequence reversed, was 23.1 mg RE / g, with a yield of 2.33%. Compared to Example 1, the absence of the pulsed electric field stage or the reversal of the treatment sequence resulted in a decrease in both the total flavonoid content and the yield, verifying the synergistic necessity of the pulsed electric field pretreatment and the sequential coupling of pulsed electric field-ultrasound-enzymatic hydrolysis in the extraction method of this invention. It is noteworthy that the yields of Comparative Examples 5 and 6 were essentially the same as those of Example 2 (material-to-liquid ratio 1:10, yield 2.32%). This indicates that even under relatively harsh extraction conditions (such as a low material-to-liquid ratio), this invention can maintain an extraction level comparable to that of schemes with partial omissions or adjustments, further confirming its process stability and applicability.

[0107] Compared with the flavonoid extract of bamboo leaves obtained by the traditional 70% ethanol extraction method in Comparative Example 1, Example 1 comprehensively surpasses it in both total flavonoid content and yield, proving that the present invention achieves superior extraction efficiency compared to organic solvent methods in a pure water system. Under the premise of no use of organic solvents and no risk of solvent residue throughout the process, it achieves flavonoid extraction efficiency equal to or even higher than that of organic solvent extraction methods, resolving the core contradiction of existing technologies where "green safety and extraction efficiency cannot be simultaneously achieved."

[0108] Compared with Example 5 (without pulsed electric field pretreatment) and Example 6 (with pulsed electric field pretreatment and ultrasonic sequence reversed), it can be seen that the yield is lower than that of Example 1 when any component is missing or the process sequence is reversed. This verifies the synergistic necessity of the pulsed electric field pretreatment and the fixed temporal coupling of pulsed electric field-ultrasound-enzymatic hydrolysis in the extraction method of the present invention.

[0109] Furthermore, the yield of Comparative Example 7 using one-year-old Phyllostachys edulis raw material was 2.18%, and the total flavonoid content was 19.2 mg RE / g, both of which were lower than those of Example 1, demonstrating the superiority of two-year-old Phyllostachys edulis raw material.

[0110] In summary, the absence of the pulsed electric field section, the change of processing sequence, and the change of bamboo leaf growth years all resulted in a significant decrease in total flavonoid content and extraction yield, confirming the synergistic effect of raw material selection and ternary segmented coupling process in this invention.

[0111] Detection Example 2 The antioxidant activity of the flavonoid extracts of bamboo leaves obtained in Examples 1-9 and Comparative Examples 1-7 was tested using the following methods: DPPH free radical scavenging rate determination: Flavonoid extracts of *Bambusa textilis* leaves prepared in Examples 1-9 and Comparative Examples 1-7 were diluted with deionized water to prepare a sample solution of 100 μg / mL (based on total flavonoids). 2 mL of the sample solution was mixed with 2 mL of 0.2 mmol / L DPPH ethanol solution, and the mixture was reacted in the dark for 30 min. The absorbance was measured at 517 nm. Vitamin C was used as a positive control (scavenging rate 94.2% at the same concentration).

[0112] Clearance rate (%) = [1-(A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0113] ABTS free radical scavenging rate determination: An equal volume of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution were mixed and reacted in the dark for 12-16 h to obtain ABTS. + The stock solution was diluted with deionized water to a absorbance of 0.70 ± 0.02 at 734 nm. 0.2 mL of the sample solution was mixed with 2 mL of ABTS. +The working solutions were mixed and reacted for 6 minutes, after which the absorbance was measured at 734 nm. Trolox was used as a positive control (scavenging rate of 92.8% at the same concentration).

[0114] The results are shown in Table 2.

[0115] Table 2 As shown in Table 2, the flavonoid extracts of *Bambusa textilis* leaves prepared in Examples 1-9 of this invention all exhibited excellent antioxidant activity. Among them, the DPPH scavenging rate of the flavonoid extract of *Bambusa textilis* leaves prepared in Example 1 reached 89.6%, and the ABTS scavenging rate reached 87.2%, significantly better than the DPPH and ABTS scavenging rates of the flavonoid extract of *Bambusa textilis* leaves prepared by pure water extraction in Comparative Example 2. It was also higher than the antioxidant activity of the flavonoid extracts of *Bambusa textilis* leaves prepared by Comparative Example 3 (using only ultrasound) and Comparative Example 4 (using only enzymatic hydrolysis), demonstrating that the pulsed electric field-ultrasound-enzymatic hydrolysis ternary coupling process of this invention causes less damage to the active structure of flavonoids.

[0116] It is worth noting that the DPPH and ABTS scavenging rates of the bamboo leaf flavonoid extract prepared in Example 1 were higher than those of the bamboo leaf flavonoid extract prepared by the conventional 70% ethanol extraction method in Comparative Example 1. This indicates that the mild extraction conditions of the present invention are more conducive to protecting the phenolic hydroxyl structure of bamboo C-glycoside flavonoids, thereby retaining a stronger free radical scavenging ability.

[0117] The antioxidant activities of the *Bambusa textilis* leaf flavonoid extracts prepared in Comparative Example 5 (without pulsed electric field pretreatment) and Comparative Example 6 (with the pulsed electric field pretreatment and ultrasound sequence reversed) were both lower than those of the *Bambusa textilis* leaf flavonoid extract prepared in Example 1. This demonstrates the importance of the integrity of the ternary temporal coupling of pulsed electric field-ultrasound-enzymatic hydrolysis in the extraction method of the present invention for preserving antioxidant activity. Furthermore, the antioxidant activity of the *Bambusa textilis* leaf flavonoid extract prepared in Comparative Example 7 using one-year-old *Bambusa textilis* raw material was significantly lower than that of the *Bambusa textilis* leaf flavonoid extract prepared in Example 1, further demonstrating the advantage of two-year-old *Bambusa textilis* raw material in terms of active ingredient accumulation.

[0118] Detection Example 3 The α-amylase and α-glucosidase inhibitory activities and inhibition kinetic parameters of the flavonoid extracts of *Bambusa textilis* leaves prepared in Examples 1-9 and Comparative Examples 1-7 were detected using the following methods: • α-Amylase inhibition rate determination: Extracts obtained by different extraction methods were prepared into sample solutions of 100 μg / mL (based on total flavonoids). 0.5 mL of the sample solution was mixed with 0.5 mL of α-amylase solution (2 U / mL), incubated at 37℃ for 10 min, then 1 mL of 1% soluble starch solution was added, and incubation continued at 37℃ for another 10 min. 2 mL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min. After cooling, the absorbance was measured at 540 nm. Acarbose was used as a positive control (inhibition rate 82.6% at the same concentration).

[0119] • α-Glucosidase inhibition rate assay: Mix 0.2 mL of sample solution with 0.2 mL of α-glucosidase solution (0.2 U / mL), incubate at 37 °C for 10 min, add 0.2 mL of 5 mmol / L p-nitrophenyl-α-D-glucopyranoside (pNPG) solution, continue incubation at 37 °C for 20 min, then terminate the reaction by adding 0.2 mL of 1 mol / L Na₂CO₃ solution, and measure the absorbance at 405 nm. Acarbose was used as a positive control (inhibition rate of 78.3% at the same concentration).

[0120] IC 50 Determination of Inhibition Type: Prepare a series of sample solutions with varying concentration gradients (10, 25, 50, 75, 100, 150, 200 μg / mL), determine the inhibition rate using the method described above, and calculate the IC50 using the concentration-inhibition rate fitting curve. 50 Values. Using the Lineweaver-Burk double reciprocal plot method, with the enzyme concentration fixed and the substrate concentration varied (0.5~10 mmol / L pNPG), the initial reaction rate was measured at different inhibitor concentrations to determine the type of inhibition.

[0121] The results are shown in Table 3.

[0122] Table 3 As shown in Table 3, the flavonoid extracts of *Bambusa textilis* leaves prepared in Examples 1-9 of this invention exhibited significant inhibitory activity against both α-amylase and α-glucosidase. The flavonoid extract of *Bambusa textilis* leaves prepared in Example 1 showed an inhibition rate of 77.8% against α-amylase and an IC50 concentration of [missing value]. 50 The concentration was 45.3 μg / mL, with an inhibition rate of 68.5% against α-glucosidase and an IC50 concentration of 45.3 μg / mL. 50 The concentration was 72.6 μg / mL, which was significantly better than the IC50 of α-amylase in the flavonoid extract of *Bambusa textilis* obtained by pure water extraction in Comparative Example 2. 50 IC50 of α-glucosidase 50 It also outperformed the IC50 of α-amylase in the flavonoid extract of *Bambusa textilis* prepared using only ultrasound in Comparative Example 3 and only enzymatic hydrolysis in Comparative Example 4.50 IC50 of α-glucosidase 50 This demonstrates that the ternary coupling process of the extraction method of the present invention is more conducive to preserving the enzyme-inhibiting activity of flavonoids.

[0123] Comparative Example 5 (without pulsed electric field pretreatment), Comparative Example 6 (with alternating pulsed electric field pretreatment and ultrasonic sequence), and Comparative Example 7 (using one-year-old *Bambusa textilis* raw materials), showed the IC50 of α-amylase in *Bambusa textilis* leaf flavonoid extract. 50 IC50 of α-glucosidase 50 All values ​​were higher than in Example 1, indicating that the absence of the pulsed electric field section, the reversal of the process sequence, and the use of one-year-old bamboo leaves as raw materials all significantly improved the IC of the extract. 50 The invention reduces the enzyme inhibition rate, confirming the necessity of the pulsed electric field-ultrasound-enzymatic hydrolysis immobilization time-coupling process in the extraction method of the present invention, which uses two-year-old bamboo leaves. The extraction method of the present invention can completely retain highly active C-glycoside flavonoids, enhance the blocking effect on key enzymes of starch decomposition, and has better potential for postprandial blood glucose regulation.

[0124] The flavonoid extracts of *Bambusa textilis* leaves prepared in Examples 1-9 of this invention exhibit competitive inhibition, which can directly bind to enzyme active sites to block starch decomposition, resulting in a more efficient sugar control mechanism. This differs from the mixed inhibition of the ethanol extract in Comparative Example 1, indicating that the active ingredient composition of the flavonoid extracts of *Bambusa textilis* leaves prepared in this invention may be unique.

[0125] Application Example 1 This application example illustrates the preparation of a herbal compound plant-based beverage composition containing flavonoids from bamboo leaves.

[0126] (1) Preparation of functional mother liquor: Take 12g of the flavonoid extract of bamboo leaves prepared in Example 1, add 600mL of purified water at 60℃, stir until completely dissolved, and pass through a 200-mesh sieve to obtain the mother liquor of flavonoid extract of bamboo leaves. Weigh out 6.0g of mulberry leaf extract, 3.6g of bitter melon extract, and 2.4g of licorice extract, add them to 600mL of purified water at 80℃, stir to dissolve, and pass through a 200-mesh sieve to obtain herbal mother liquor; Weigh out 480g of erythritol, 2.4g of steviol glycosides and 24g of citric acid, add them to 800mL of purified water and stir until completely dissolved to obtain a sweet and sour liquid.

[0127] (2) Blending and homogenization: Add the mother liquor of flavonoid extract of bamboo leaves, herbal mother liquor and sweet and sour liquid obtained in step (1) to the blending tank in sequence, add xanthan gum accounting for 0.02% of the total mass of the herbal compound plant-based beverage composition, and make up to 12L with purified water; turn on the stirrer, stir evenly, adjust the pH to 4.2 with citric acid, preheat the mixture to 65℃, and homogenize it under 25MPa pressure.

[0128] (3) Sterilization and filling: The homogenized liquid in step (2) is pasteurized at 85°C for 15 minutes, hot-filled (temperature ≥ 85°C) into PET bottles, sealed immediately, and rapidly cooled to room temperature (15~30°C) to obtain a herbal compound plant-based beverage composition containing flavonoids from bamboo leaves, denoted as Y1.

[0129] Comparative Example 8 The method of Application Example 1 was followed, except that no herbal mother liquor was added, and other conditions remained the same, to obtain a plant-based beverage composition containing flavonoids from bamboo leaves, denoted as D8.

[0130] Detection Example 4 The herbal compound plant-based beverage composition containing bamboo leaf flavonoids prepared in Example 1 and the plant-based beverage composition containing bamboo leaf flavonoids prepared in Comparative Example 8 were subjected to an in vitro simulated digestion experiment. The specific method is as follows: The in vitro digestive model was designed with reference to the 2020 edition of the Chinese Pharmacopoeia and commonly used international methods, simulating the digestive process in the oral cavity, stomach, and small intestine.

[0131] Oral digestion: Take 10 mL of sample solution, add α-amylase solution (final concentration 75 U / mL), adjust pH to 6.8 with 1 mol / L HCl or NaOH, and shake at 37℃ for 2 min.

[0132] Gastric digestion: Transfer the oral digestive fluid to an Erlenmeyer flask, adjust the pH to 2.0 with 1 mol / L HCl, add pepsin solution (final concentration 2000 U / mL), and shake the reaction at 37℃ for 2 h.

[0133] Small intestinal digestion: Adjust the pH of gastric digestive juice to 6.8 with 1 mol / L NaHCO3, add pancreatic enzyme solution (final concentration 100 U / mL) and α-glucosidase solution (final concentration 2 U / mL), and add soluble starch (final concentration 1%, w / v). Shake and react at 37℃ for 2 h, and take samples at 0, 20, 60 and 120 min respectively.

[0134] • Glucose release determination: The DNS (3,5-dinitrosalicylic acid) colorimetric method was used. 1 mL of sterilizing solution was added to 2 mL of DNS reagent, the mixture was boiled in a water bath for 5 min, cooled, and the absorbance was measured at 540 nm. Quantification was performed using a glucose standard curve.

[0135] Starch hydrolysis degree (%) = (glucose release × 0.9 / total starch content) × 100%.

[0136] • Formula for calculating the estimated glycemic index (eGI): eGI = 0.862 × area under the hydrolysis curve (sample) / area under the hydrolysis curve (white bread control) × 100. The eGI value for the white bread control was set to 100.

[0137] • The positive control was acarbose (final concentration 50 μg / mL).

[0138] The results are shown in Table 4.

[0139] Table 4 As shown in Table 4, the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Example 1 of this invention had a starch hydrolysis degree of 47.6% at the 120-minute small intestinal digestion endpoint, which was significantly lower than the starch hydrolysis degree of the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8, with a decrease of 14.7%. The herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Example 1 of this invention also had a glucose release of 22.0 mg / g starch at the 120-minute small intestinal digestion endpoint, which was 15.4% lower than the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8. The herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Example 1 of this invention had an estimated GI value (eGI) of 55.2 at the 120-minute small intestinal digestion endpoint, falling into the low-to-medium GI food category (≤55~60). In contrast, the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids in Comparative Example 8, which did not contain herbal mother liquor, had an eGI value of 63.5, belonging to the medium-to-high GI food category. This demonstrates that the combination of *Bambusa textilis* leaf flavonoids with mulberry leaf and bitter melon extracts has a significant synergistic inhibitory effect on starch hydrolysis, and can significantly delay the glucose release rate during digestion.

[0140] Compared with the positive control acarbose, the blood sugar lowering effect of the beverage of this invention is slightly lower than that of acarbose. However, it belongs to a natural food and medicine homology compound system, has no side effects of chemical blood sugar lowering drugs, and has the advantage of large-scale food application. The blank digestive system shows rapid starch decomposition and large release of glucose, which further verifies that the beverage can effectively block the decomposition and absorption of carbohydrates and achieve the effect of stabilizing postprandial blood sugar.

[0141] Comparing the digestion time points, at 20 min, 60 min, and 120 min, the starch hydrolysis degree and glucose release of the herbal compound plant-based beverage composition containing bamboo leaf flavonoids prepared in Example 1 of this invention were consistently lower than those of the plant-based beverage composition containing bamboo leaf flavonoids without herbal mother liquor in Comparative Example 8. This indicates that the synergistic sugar control effect of the compound components lasts throughout the entire digestion cycle, effectively inhibits sugar decomposition, and has stable postprandial blood glucose regulation potential.

[0142] Case 5 The storage stability of the herbal compound plant-based beverage composition containing flavonoids from bamboo leaves prepared in accordance with Example 1 was tested, and the specific method is as follows: The herbal compound plant-based beverage composition containing flavonoids from bamboo leaves prepared in Application Example 1 was stored in a refrigerator at 4°C, a room temperature at 25°C, and a constant temperature incubator at 37°C. Samples were taken and tested on days 0, 7, 14, 30, and 60.

[0143] • Flavonoid retention rate: The total flavonoid content on storage day 0 was taken as 100%, and the result was determined by the NaNO2-Al(NO3)3-NaOH colorimetric method. The result is expressed as "mean ± SD (n=3)".

[0144] • Turbidity: Measured using a portable turbidimeter, unit NTU (turbidity scattering turbidity unit).

[0145] • Precipitation volume: Take 50 mL of sample, centrifuge at 4000 rpm for 10 min, discard the supernatant, dry the precipitate at 105℃ to constant weight and weigh it.

[0146] • Color L value: Measured using a colorimeter, the L value represents the brightness (0 = black, 100 = white).

[0147] The results are shown in Table 5.

[0148] Table 5 Table 6 As can be seen from the data in Tables 5 and 6, the storage temperature and storage time directly affect the physicochemical stability, flavonoid retention level, antioxidant activity and sensory quality of the herbal compound plant-based beverage composition containing flavonoids from bamboo leaves prepared in Example 1 of this invention. Low temperature storage can significantly delay product deterioration.

[0149] After 30 days of storage at 4°C, the herbal compound plant-based beverage composition containing flavonoids from bamboo leaves prepared in Example 1 of this invention showed only a slight increase in turbidity and no visible precipitation. The flavonoid retention rate reached 95.2%, and the antioxidant activity of DPPH and ABTS decreased only slightly. After 60 days of storage, there was no precipitation, the flavonoid retention rate was still higher than 92%, the overall sensory score was 8.2 points, the bitterness score was only 2.8 points, the consumer acceptability reached 90%, and the product's clear appearance and core functional activity could be maintained for a long time in a low-temperature environment.

[0150] The stability at room temperature (25℃) was the second lowest. After 30 days of storage, a small amount of precipitation began to appear, the flavonoid retention rate dropped to 89.6%, the antioxidant activity decreased significantly, the bitterness intensified, and the overall sensory score dropped to 7.8. Under the accelerated storage conditions at 37℃, the product deteriorated significantly faster. After 30 days of storage, the turbidity increased significantly, the amount of precipitation increased significantly, the flavonoid retention rate was only 82.3%, the antioxidant activity decreased significantly, the bitterness reached 5.2, and the palatability was greatly reduced. This proves that high temperature accelerates flavonoid oxidation, system stratification, and flavor deterioration.

[0151] The herbal compound plant-based beverage composition of the present invention, containing flavonoids from bamboo leaves, with the addition of 0.01-0.05% xanthan gum stabilizer and pH controlled at 4.0-4.2, can effectively inhibit the aggregation and precipitation of flavonoid polyphenols during storage. Combined with erythritol-stevioside compound sweetener, it can mask the inherent bitterness of flavonoids for a long time. Comprehensive physicochemical, activity, and sensory data show that this beverage is suitable for refrigeration or short-term storage at room temperature. Refrigeration at 4°C can ensure a stable shelf life of at least 1 month, which has the storage performance advantages of industrial beverages.

[0152] Case 6 The herbal compound plant-based beverage composition containing bamboo leaf flavonoids prepared in Example 1 and the plant-based beverage composition containing bamboo leaf flavonoids prepared in Comparative Example 8 were subjected to sensory evaluation. The specific methods are as follows: A professional sensory evaluation team of 10 people (5 men and 5 women, aged 22-45) was organized to conduct blind tests. All evaluators had received basic sensory training and were able to identify and describe basic sensory attributes such as color, aroma, and taste.

[0153] Sensory rating criteria: Color (2 points maximum): 1.5 points or above is light yellow-green, clear and bright; 1.0~1.5 points is dark or slightly cloudy; below 1.0 points is obviously cloudy or has abnormal color.

[0154] Aroma (out of 2 points): 1.5 points or above indicates a clear bamboo leaf or herbal aroma with no off-odors; 1.0 to 1.5 points indicates a faint aroma or a slight off-odor; below 1.0 points indicates an unpleasant aroma or a noticeable unpleasant odor.

[0155] Smoothness of mouthfeel (out of 3): 2.5 points or above means smooth and without roughness; 2.0 to 2.5 points means relatively smooth with a slight grainy texture; below 2.0 points means rough or with obvious grainy texture.

[0156] Sweet and sour palatability (out of 3 points): 2.5 points or above indicates a balanced sweet and sour ratio and good palatability; 2.0 to 2.5 points indicates a basically balanced sweet and sour ratio with a slight tendency to be too sour or too sweet; below 2.0 points indicates an unbalanced sweet and sour ratio and poor palatability.

[0157] • Bitterness rating criteria: A 10-point scale is used for quantitative scoring, where 1 point = almost no bitterness and 10 points = extremely bitter / extremely astringent. Evaluators make a comprehensive assessment based on the intensity and persistence of the bitterness sensation in the mouth.

[0158] The results are shown in Table 7.

[0159] Table 7 As shown in Table 7, the overall sensory score of the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Application Example 1 was 8.7 / 10, significantly better than that of the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8. The difference between the two was statistically significant (P<0.05). Specifically, the bitterness score of the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8 was as high as 4.2 / 10, while the bitterness score of the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Application Example 1 was only 2.3 / 10, a decrease of 45.2%. This indicates that the combination of mulberry leaves, bitter melon, and licorice, as well as the compound sweetener of erythritol and steviol glycosides, can effectively mask the bitterness of beverages with high flavonoid content. Glycyrrhizin in licorice has a natural sweetness masking effect, while the fragrance of mulberry leaves and the aftertaste of bitter melon can neutralize the bitterness of flavonoids from a flavor perspective.

[0160] Furthermore, the aroma score of the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Example 1 (1.7 / 10) was significantly higher than that of the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8 (1.2 / 10), indicating that the addition of herbal extracts imparts a richer aroma profile to the beverage. The sensory evaluation panel generally described the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids prepared in Example 1 as having a "light yellow-green color, clear and bright appearance, a fresh bamboo leaf aroma and a complex herbal fragrance, a pleasantly sweet and sour taste, a smooth mouthfeel, almost no bitterness, and a clean aftertaste," while the plant-based beverage composition containing *Bambusa textilis* leaf flavonoids without herbal mother liquor in Comparative Example 8 was described as having a "dark color, a monotonous bamboo leaf flavor, a noticeable bitterness, and a thin mouthfeel." These results demonstrate that the herbal compounding scheme of the herbal compound plant-based beverage composition containing *Bambusa textilis* leaf flavonoids of the present invention has significant advantages in improving the sensory quality of beverages and solves the common bitterness problem in beverages with high flavonoid content. The flavonoid extract from bamboo leaves prepared by this invention can be used as a general-purpose food additive in the form of a concentrated liquid (30-40% solids content) or spray-dried powder, and can be widely applied in foods such as jellies, yogurts, nutrition bars, biscuits, and functional candies at an addition amount of 0.1-1.0% (w / w). After addition, the products retain their original flavor and texture characteristics, while also providing them with dual effects of anti-oxidation and blood sugar control. Specific preparation methods for the above-mentioned foods can employ conventional processes in the field, and will not be listed individually here.

[0161] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0162] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0163] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for extracting flavonoids from bamboo leaves of *Bambusa textilis*, characterized in that, The extraction method includes the following steps: mixing bamboo leaf powder with pure water, and sequentially subjecting it to pulsed electric field treatment, ultrasonic treatment, and enzymatic hydrolysis; after enzymatic hydrolysis, performing enzyme inactivation treatment; separating solid and liquid, collecting the supernatant to obtain bamboo leaf flavonoid extract; The bamboo leaf powder mentioned is from two-year-old bamboo leaves.

2. The extraction method according to claim 1, characterized in that, The ratio of the bamboo leaf powder to pure water is 1g:10~30mL.

3. The extraction method according to claim 1 or 2, characterized in that, The conditions for pulsed electric field processing are: electric field strength 10~50kV / cm, pulse width 10~50μs, pulse frequency 50~300Hz, and effective processing time 50~300μs. Preferably, the conditions for pulsed electric field processing are: electric field strength 20~35kV / cm, pulse width 20~30μs, and pulse frequency 100~200Hz; More preferably, the pulsed electric field treatment is followed by a resting period of 5-15 minutes before the ultrasonic treatment is performed.

4. The extraction method according to any one of claims 1-3, characterized in that, The conditions for ultrasonic treatment are: temperature 15~30℃, ultrasonic power 300~500W, frequency 40kHz, and ultrasonic time 10~30min. Preferably, the conditions for ultrasonic treatment are: ultrasonic power 350~450W, ultrasonic time 15~20min.

5. The extraction method according to any one of claims 1-4, characterized in that, The enzymatic hydrolysis process is as follows: after ultrasonic treatment, the pH is adjusted to 4.5-5.5 using a pH adjuster, cellulase is added, and enzymatic hydrolysis is carried out at a temperature of 45-55℃ for 45-90 minutes. The pH adjuster is selected from one or more of citric acid, malic acid, lactic acid, and tartaric acid; The amount of cellulase added is 0.5~2.5% of the mass of the bamboo leaf powder, and the enzyme activity is ≥400U / mg.

6. The extraction method according to any one of claims 1-5, characterized in that, The solid-liquid separation is performed by centrifugation or vacuum filtration. The extraction method comprises ultrasonic treatment, enzymatic hydrolysis, enzyme inactivation, and solid-liquid separation, forming an extraction cycle. The extraction cycle is repeated 2 to 3 times. In each extraction cycle, the ratio of filter residue to pure water is 1g:10 to 30mL. The total time for ultrasonic treatment and enzymatic hydrolysis is 60 to 110min. The extraction method further includes concentrating the extract of flavonoids from bamboo leaves under reduced pressure at a temperature of 50-55℃ and a pressure of -0.10--0.08MPa to 0.2-0.5 times the original volume to obtain a concentrated extract of flavonoids from bamboo leaves. Preferably, the concentrated flavonoid solution of bamboo leaves is spray-dried to obtain bamboo leaf flavonoid extract powder; The spray drying conditions include: inlet air temperature of 160~180℃, outlet air temperature of 80~90℃, and feed rate of 40~60 mL / min.

7. The flavonoid extract of bamboo leaves prepared by the extraction method according to any one of claims 1-6.

8. A plant-based beverage composition, characterized in that, The plant-based beverage composition comprises flavonoid extracts from bamboo leaves, mulberry leaves, bitter melon extract, licorice extract, sweeteners, acidulants, stabilizers, and water; The flavonoid extract of bamboo leaves from *Ciba fasciata* is the same as that described in claim 7.

9. The plant-based beverage composition according to claim 8, characterized in that, The sweetener is a mixture of erythritol and steviol glycosides, wherein the mass ratio of erythritol to steviol glycosides is 10-20:1; The acidulant is selected from one or more of citric acid, malic acid, lactic acid and tartaric acid; The stabilizer is selected from one or more of xanthan gum, sodium carboxymethyl cellulose, sodium alginate, and carrageenan; Preferably, the pH of the plant-based beverage composition is 3.5-5.0, more preferably 4.0-4.2; Preferably, per 1000 mL of beverage, the mass-volume percentage of each component in the plant-based beverage composition is as follows: 0.1-2.0% of *Bambusa textilis* leaf flavonoid extract, 0.2-1.5% of mulberry leaf extract, 0.1-1.0% of bitter melon extract, 0.1-0.5% of licorice extract, 3.0-8.0% of erythritol, 0.01-0.05% of steviol glycosides, 0.1-0.3% of citric acid, 0.01-0.05% of stabilizer, and the remainder being purified water; Preferably, the mass-volume percentage of each component in the plant-based beverage composition per 1000 mL of beverage is as follows: 1.0% of bamboo leaf flavonoid extract, 0.5% of mulberry leaf extract, 0.3% of bitter melon extract, 0.2% of licorice extract, 4.0% of erythritol, 0.02% of steviol glycosides, 0.2% of citric acid, 0.02% of stabilizer, and the remainder is purified water.

10. The use of the flavonoid extract of *Bambusa textilis* leaves as described in claim 7 or the plant-based beverage composition as described in claim 8 or 9 in the preparation of food, characterized in that, The flavonoid extract of bamboo leaves from *Bambusa textilis* is added to food as a food additive in the form of a concentrated liquid or powder, at a concentration of 0.1-1.0% of the total mass of the food. The food product is selected from one or more of the following: jelly, yogurt, nutrition bars, biscuits, or functional candies.

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