An antifungal flavonoid component from discarded leaves of water bamboo, its preparation method and uses

Abstract

This invention relates to the field of agricultural waste utilization technology, specifically to an antifungal flavonoid component from waste leaves of water chestnut, its preparation method, and its uses. The defatting combined with flash / enzymatic extraction method provided by this invention can efficiently prepare flavonoids from waste leaves of water chestnut; a flavonoid component with highly efficient antifungal activity can be isolated from the flavonoids prepared by this method; the minimum inhibitory concentration (MIC) of the flavonoid component from waste leaves of water chestnut against Candida albicans is 1.25 mg / mL; this flavonoid component can be used to prepare antifungal sprays.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural waste utilization, and particularly relates to an antifungal flavonoid component from waste leaves of water bamboo, a preparation method thereof, and uses thereof. Background Art

[0002] Water bamboo, alias: high melon, water bamboo shoot, belongs to the genus Zizania of the family Poaceae, and is a perennial aquatic and rhizomatous herbaceous plant. It is native to China and Southeast Asia, and is planted in provinces such as Hebei, Jiangsu, Zhejiang, Anhui, Jiangxi, Hunan, Taiwan, Hubei, and Hainan in China. Water bamboo is a plant with both medicinal and edible properties, and its edible part is its enlarged fleshy stem. As food, water bamboo has high nutritional value. It not only contains rich proteins, fats, carbohydrates, vitamins, and minerals, but also contains various essential amino acids for the human body. As a medicinal material, water bamboo is sweet and slightly cold in taste, and has the effects of removing heat, promoting fluid production, quenching thirst, diuretic, dehumidifying, and dredging. Therefore, it has the reputation of "ginseng in water". The water bamboo plant is tall and has a high biological yield. The biomass of the water bamboo bracts accounts for 50% - 70% of the total mass of the water bamboo plant. For double-season water bamboo, the fresh bracts produced in the current year are more than 5000 kg per 667 m ,

[0005] ,

[0004] , , above.

[0003] Flavonoids are a class of plant secondary metabolites that are widely present in plants. They not only have a large number and variety of species, but also have complex and diverse structural types. They can not only participate in various activities in cells in the body, but also have a variety of pharmacological effects. It is reported that appropriate intake of flavonoids can reduce the incidence of diseases such as cancer, tumors, cardiovascular diseases, lipid peroxidation, and osteoporosis. In addition, plant flavonoids also have a variety of antibacterial effects, including inhibiting the growth of bacteria, fungi, and viruses. They can play a role by destroying the cell membrane structure of bacteria, interfering with the biosynthesis process of bacteria, inhibiting the biosynthesis process of viruses, and inhibiting the replication of viruses. These studies provide a sufficient theoretical basis for their application in the fields of medicine and food, and accelerate the development and utilization of flavonoids. There are many extraction methods for plant active flavonoids, and the most common one is the solvent extraction method. In addition, the synergistic effect of some auxiliary means can effectively improve the yield of flavonoids, such as enzymatic hydrolysis, microwave, ultrasonic, and flash extraction technologies. However, there has been no report on the antifungal flavonoids from waste leaves of water bamboo so far. Therefore, it is necessary to conduct research on this to solve the problem of recycling waste leaves of water bamboo and provide a basis for the comprehensive and high-value development of water bamboo resources.

[0004] In summary, the present invention provides an antifungal flavonoid component from waste leaves of water bamboo, a preparation method thereof, and uses thereof, providing a method and approach for the high-value utilization of water bamboo bracts and even similar agricultural waste resources. Summary of the Invention

[0005] The purpose of this invention is to provide an antifungal flavonoid component from waste leaves of water bamboo, its preparation method, and its uses. Compared with traditional extraction methods, the defatting combined with flash / enzymatic extraction method provided by this invention can efficiently prepare flavonoids from waste leaves of water bamboo. Flavonoid components with highly efficient antifungal activity can be isolated from the flavonoids from waste leaves of water bamboo prepared by this method.

[0006] According to a first aspect of the present invention, the present invention provides a method for preparing flavonoid components from waste leaves of water chestnut, wherein waste leaf powder of water chestnut is defatted, soaked in deionized water, then subjected to cell wall disruption treatment using a flash cell disruptor, then a certain amount of cellulase is added for enzymatic hydrolysis, and finally extracted with a certain concentration of ethanol aqueous solution for a certain period of time; crude flavonoids are obtained by concentration, centrifugation, and freeze drying.

[0007] Preferably, the method for preparing the flavonoid component from waste water chestnut leaves specifically includes the following steps:

[0008] Fresh water bamboo husks were washed, dried to constant weight, pulverized, and passed through a 40-mesh sieve. A certain amount of the water bamboo husk powder was placed in a Soxhlet extractor and defatted by reflux in a water bath with petroleum ether (boiling range 60–90℃) to obtain defatted water bamboo husk powder. The defatted water bamboo husk powder was placed in a beaker, and a certain amount of deionized water was added. Flash extraction was performed for a certain time using a flash mixer under a certain voltage. After the extraction, the pH of the extract was adjusted to 5.0 ± 0.2. Then, a certain amount of cellulase was added, and the extract was enzymatically hydrolyzed at 37℃ for a certain time. The enzyme was then inactivated in a 90℃ water bath for 10 minutes. Anhydrous ethanol and deionized water were added to adjust the final ethanol concentration of the extract to 60% (V%). The extract was then further extracted at 60℃ for 60 minutes. The extract was concentrated, centrifuged, and freeze-dried to obtain the crude flavonoid fraction.

[0009] Preferably, the flash extraction voltage is 100V to 150V, more preferably 110V;

[0010] Preferably, the flash extraction time is 1 min to 8 min, more preferably 4.1 min;

[0011] Preferably, the enzymatic hydrolysis time is 40 min to 90 min, more preferably 60 min;

[0012] Preferably, the enzyme dosage is 0.3% to 0.8% (mass ratio, i.e., the mass of defatted water chestnut waste leaf powder), more preferably 0.7%;

[0013] Preferably, the Flash extraction voltage is 110V, the Flash extraction time is 4.1min, the enzymatic hydrolysis time is 60min, and the enzyme dosage is 0.7%. Under these conditions, the crude flavonoid extraction rate of waste leaves of water chestnut is 2.21±0.16%.

[0014] The preparation method of the flavonoid component described in this invention further includes the separation and purification of the flavonoid component from waste water chestnut leaves. Specifically, 20g of activated DM301 macroporous resin is wet-packed into a column (inner diameter 16mm × length 300mm). The loading solution concentration is 0.1mg / mL, the loading volume is 30mL, the loading rate is 1.0 BV / h, the eluent is anhydrous acetone, the elution rate is 2.0 BV / h, and the eluent volume is 60mL. The effluent is concentrated under reduced pressure and freeze-dried under vacuum to obtain the flavonoid component from waste water chestnut leaves. The flavonoid content was determined to be 56.23±3.28% by the NaNO2-Al(NO3)3 method.

[0015] According to a second aspect of the invention, the use of the waste flavonoid component of *Zizania latifolia* in antifungal products is provided; it can be used to prepare antifungal sprays.

[0016] The present invention has the following beneficial effects:

[0017] 1) The defatting combined flash / enzyme extraction method provided by the present invention has significant advantages in improving the yield of flavonoids from waste leaves of water chestnut and is expected to be applied to the efficient extraction of plant active factors.

[0018] 2) This invention optimizes the purification method for crude flavonoids from waste leaves of water chestnut, providing a minimum inhibitory concentration (MIC) of 1.25 mg / mL against Candida albicans. This can be used to prepare antifungal products. Attached Figure Description

[0019] Figure 1 Effect of flash voltage on the yield of crude flavonoids from waste leaves of wild rice;

[0020] Figure 2 Effect of flash time on the yield of crude flavonoids from waste leaves of wild rice;

[0021] Figure 3 Effect of enzyme dosage on the yield of crude flavonoids from waste leaves of water chestnut;

[0022] Figure 4 Effect of enzymatic hydrolysis time on the yield of crude flavonoids from waste leaves of water chestnut;

[0023] Figure 5 The yield of crude flavonoids by two extraction methods. Detailed Implementation

[0024] Waste leaves of water bamboo were collected in June 2023 from riverbanks surrounding the Southeast Development Zone of Changshu City, Jiangsu Province; the flash extraction controller was provided by Xi'an Taikang Biotechnology Co., Ltd., model JHBE-50T; Candida albicans was purchased from China General Microbiological Culture Collection Center, number: CGMCC2.4159; all chemical reagents were purchased from Shanghai Sangon Biotech Co., Ltd.; all experiments were performed in triplicate, and data are expressed as mean ± SD. Statistical analysis of data was performed using t-test or ANOVA, and P < 0.05 was considered statistically significant.

[0025] Example 1

[0026] Fresh water bamboo buds were washed, dried to constant weight, pulverized, and passed through a 40-mesh sieve. A certain amount of the water bamboo bud powder was placed in a Soxhlet extractor and defatted by reflux in a water bath with petroleum ether (boiling range 60–90℃). The petroleum ether was then discarded and evaporated. The defatted water bamboo bud powder was placed in a beaker, and 40 times its weight of deionized water was added. Flash extraction was performed for a certain time using a flash mixer under a certain voltage. After extraction, the pH of the extract was adjusted to 5.0 ± 0.2. Then, a certain amount of cellulase was added, and the extract was enzymatically hydrolyzed at 37℃ for a certain time. The enzyme was then inactivated in a 90℃ water bath for 10 minutes. Anhydrous ethanol and deionized water were added to adjust the final ethanol concentration of the extract to 60%. The extract was then further extracted at 60℃ for 60 minutes. The extract was concentrated, centrifuged, and freeze-dried to obtain crude flavonoids.

[0027] 1.1 Single-factor experiment

[0028] Process parameters: Following the method in Example 1, the initial settings for each process parameter are as follows: flash extraction voltage 100V, flash extraction time 1min, enzyme dosage 0.4% (i.e., 0.004g cellulase added per g of defatted water chestnut waste leaf powder), and enzymatic hydrolysis time 50min.

[0029] 1.1.1 Effect of flash voltage on the yield of crude flavonoids from waste leaves of water chestnut

[0030] In investigating the effect of flash voltage on the yield of crude flavonoids from waste leaves of wild rice, the flash voltage was varied while other factors were kept constant. The results are as follows: Figure 1 As shown.

[0031] Depend on Figure 1 It was found that when the extraction voltage was between 100V and 110V, the yield of crude flavonoids from waste leaves of water chestnut showed an increasing trend, reaching its highest level at 110V, after which the yield began to decline. The reason for this is likely that as the extraction voltage increases, frictional heat is generated among the components inside the flash extractor, leading to a rapid increase in temperature and the destruction of flavonoids, resulting in a decrease in yield. Therefore, the optimal extraction voltage was determined to be 110V.

[0032] 1.1.2 Effect of flash time on the yield of crude flavonoids from waste leaves of water chestnut

[0033] In investigating the effect of flash time on the yield of crude flavonoids from waste leaves of wild rice, the flash time was varied while other factors were kept constant. The results are as follows: Figure 2 As shown.

[0034] Depend on Figure 2 It was found that the yield of crude flavonoids from waste leaves of water chestnut increased when the extraction time was between 1 and 4 minutes, reaching its highest level at 4 minutes, after which the yield began to decline. This is likely because extending the flash extraction time causes a rapid increase in the internal temperature of the extractor, destroying flavonoids and thus reducing the yield. Therefore, the optimal extraction time was determined to be 4 minutes.

[0035] 1.1.3 Effect of enzyme dosage on the yield of crude flavonoids from waste leaves of water chestnut

[0036] In investigating the effect of enzyme dosage on the yield of crude flavonoids from waste leaves of water chestnut, the enzyme dosage was varied while other factors were kept constant. The results are as follows: Figure 3 As shown.

[0037] Depend on Figure 3 It can be seen that as the enzyme dosage increases, the yield of flavonoids from waste water chestnut leaves initially rises and then falls, with the highest yield observed when the enzyme dosage is 0.7%. This is likely because as the enzyme dosage increases, the cellulose structure is disrupted, promoting the dissolution of its contents. Once the enzyme concentration in the solution reaches saturation, further increasing the dosage will block the flavonoid dissolution channels, and increasing the enzyme dosage can also cause competition or even inhibition between enzymes. Therefore, the optimal cellulase dosage is 0.7%.

[0038] 1.1.4 Effect of enzymatic hydrolysis time on the yield of crude flavonoids from waste leaves of water chestnut

[0039] In investigating the effect of enzymatic hydrolysis time on the yield of crude flavonoids from waste leaves of water chestnut, the hydrolysis time was varied while other factors were kept constant. The results are as follows: Figure 4 As shown.

[0040] Depend on Figure 4 It can be seen that the flavonoid yield is highest when the enzymatic hydrolysis time is 60 min. This is because if the hydrolysis time is too short, the reaction between the enzyme and the substrate is insufficient, resulting in a lower yield. Furthermore, as the hydrolysis time increases, the flavonoid yield no longer increases. When the enzyme is sufficient, extending the hydrolysis time can lead to the degradation of flavonoids, actually decreasing the yield. Therefore, the optimal hydrolysis time is 60 min.

[0041] 1.2 Response Surface Experiment

[0042] Based on the single-factor experiments, the Box-Behnken method in Design-Expert V8.0.6.1 software was used to design experiments. Four factors were used: flash voltage, flash time, enzyme dosage, and enzymatic hydrolysis time. Each factor was set to three levels: high (1), medium (0), and low (-1) (Table 1). The design scheme and results are shown in Table 2, and the results of the analysis of variance are shown in Table 3.

[0043] Table 1 Plackett-Burman Design Factor Levels

[0044]

[0045] Table 2. Experimental combinations and results of the Box-Behnken method design.

[0046]

[0047]

[0048] Through multiple regression fitting, the quadratic regression model equation of the predicted value of Y on the encoded values ​​of A, B, C, and D is obtained:

[0049] Y=2.220+0.028A-0.008B+0.038C-0.110D+0.033A B-0.003A C+

[0050] 0.013A D+0.008B C-0.070B D-0.048C D-0.297A2-0.376B2-0.327C2

[0051] -0.216D2.

[0052] Table 3. Results of ANOVA for the regression model

[0053]

[0054] Note: R 2 =0.9657, Adj R 2 =0.9315, Pred R 2 =0.9052; Significance: * indicates a significant difference, P<0.05; ** indicates an extremely significant difference, P<0.01; ns indicates no significant difference, P>0.05.

[0055] Table 3 shows that the p-value of this model is <0.0001, indicating that the selected model is highly significant; the coefficient of determination R0 is... 2 =0.9657, indicating that 96.57% of the change in the response value comes from the selected variable; R 2 adj=0.9315, meaning that the model can explain 93.15% of the variability in the experimental data. Analysis of variance of the lack-of-fit term shows that the lack-of-fit term is not significant (P=0.1251>0.05), indicating that the model is stable and can predict the actual changes in the yield of flavonoids from waste water chestnut leaves relatively well.

[0056] Analysis using Design-Expert software revealed the following conditions for preparing flavonoids from waste water chestnut leaves using a defatting combined flash / enzymatic extraction method: enzyme dosage 0.704%, hydrolysis time 60.161 min, flash time 4.077 min, and flash voltage 107.343 V. Under these conditions, the model predicted a flavonoid yield of 2.239%. Based on the software prediction results and the feasibility of the actual process settings, an enzyme dosage of 0.7%, hydrolysis time of 60 min, flash time of 4.0 min, and flash voltage of 107 V were selected as the extraction conditions for process verification. The experiment verified that the actual yield of flavonoids from waste water chestnut leaves was 2.21 ± 0.16%, with a relative error of 1.30% (< 5.0%) compared to the theoretical prediction. This indicates that the optimized extraction process for flavonoids from waste water chestnut leaves based on this response surface methodology is effective and feasible.

[0057] Comparative Example

[0058] Based on the optimal process obtained in Example 1, in order to demonstrate that the defatting combined with flash / enzymatic extraction of flavonoids from waste leaves of wild rice provided by this invention is superior to the enzyme-synergistic ultrasound-assisted alcohol extraction method in the applicant's authorized invention patent ZL202210693182.X, this invention further conducted comparative experiments:

[0059] Option A: Enzyme-assisted ultrasound-assisted extraction method

[0060] The collected fresh water chestnut bracts were washed, dried to constant weight, pulverized, and passed through a 40-mesh sieve. A certain amount of water chestnut bract powder was soaked in disodium hydrogen phosphate-citric acid buffer (pH=5, solid-liquid ratio 1g / 40mL), and then 0.7% cellulase was added. After enzymatic hydrolysis at 37℃ for 60min, the enzyme was inactivated in a 90℃ water bath for 10min. Then, a certain amount of anhydrous ethanol and deionized water were added to adjust the final ethanol concentration of the system to 60%. Finally, the mixture was ultrasonically treated at 55℃ and ultrasonic power of 704W for 50min, concentrated, centrifuged, and freeze-dried to obtain crude flavonoids.

[0061] The results showed that the yield of flavonoids prepared by the enzyme-assisted ultrasound-assisted extraction method was 1.92 ± 0.12%, which was lower than that of the method in the embodiments of the present invention. Figure 5 This indicates that defatting combined with flash / enzymatic extraction is more effective in promoting the release of flavonoids from waste leaves of water chestnut.

[0062] Example 2

[0063] DM301 macroporous resin was soaked in anhydrous ethanol for 24 hours at room temperature, rinsed with distilled water until no alcohol odor remained, soaked in 5% NaOH solution for 5 hours, washed with distilled water until neutral, and finally soaked in 5% HCl solution for 5 hours, washed with distilled water until neutral. 20g of the treated DM301 macroporous resin was wet-packed into a column (16mm inner diameter × 300mm length). The loading solution concentration was 0.1mg / mL (crude flavonoids were dissolved with a small amount of 60% ethanol before adding distilled water), the loading volume was 30mL, the loading rate was 1.0 BV / h, the eluent was anhydrous acetone, the elution rate was 2.0 BV / h, and the eluent volume was 60mL. The eluent was concentrated under reduced pressure and freeze-dried under vacuum to obtain the flavonoid fraction from waste water chestnut leaves. The flavonoid content was determined to be 56.23±3.28% by the NaNO2-Al(NO3)3 method.

[0064] Comparative Example

[0065] Based on Example 2, in order to demonstrate that the yield of flavonoid components from waste water chestnut leaves provided by this invention is superior to the yield of flavonoid components eluted at other concentrations, the following comparative experiment was conducted:

[0066] Scheme B: Determination of the yield of flavonoid components obtained by elution with 60%–90% acetone solution. Take 20g of treated DM301 macroporous resin and pack it into a column (inner diameter 16mm × length 300mm). The concentration of the loading solution is 0.1mg / mL (the crude flavonoids are dissolved with a small amount of 60% ethanol and then distilled water is added). The loading volume is 30mL and the loading speed is 1.0BV / h. The eluents are 60%, 70%, 80%, and 90% acetone solutions, respectively. The elution speed is 2.0BV / h and the eluent volume is 60mL. The effluent is concentrated under reduced pressure and freeze-dried under vacuum to obtain the flavonoid components FC1, FC2, FC3, and FC4 from the waste leaves of water chestnut. The flavonoid content determined by the NaNO2-Al(NO3)3 method was 32.48±3.25%, 38.32±3.25%, 40.36±2.95%, and 42.79±6.13%, all of which were lower than the flavonoid content in the flavonoid fraction obtained by elution with anhydrous acetone.

[0067] Example 3: Determination of the antifungal activity of flavonoid components from waste water chestnut leaves

[0068] Weigh 2.0g of the flavonoid component from the waste leaves of water bamboo (i.e., the one prepared in Example 2), dissolve it with a small amount of 60% ethanol, and then add distilled water to make the final concentration 20mg / mL. Add 100 μL of sterile PDA liquid medium to wells 2-10 of a sterile 96-well plate. Add 100 μL of a prepared 20 mg / mL solution of flavonoids from waste leaves of *Zizania latifolia* to wells 1 and 2. Mix well 2, then add 100 μL of this solution to well 3. Mix well 3, then add 100 μL of this solution to well 4. Continue diluting by two-fold until well 10 is reached. After mixing, discard 100 μL of this solution. Add 100 μL of bacterial suspension (*Candida albicans* suspension in logarithmic growth phase) to wells 1-10. This results in final flavonoid concentrations of 10000, 5000, 2500…39.0625, 19.53125 μg / mL in wells 1-10 of the 96-well plate, respectively. Each treatment is repeated three times. Immediately afterward, measure the OD of each well using a microplate reader. 600 After recording the data, the 96-well plate was placed in a 37°C constant temperature and humidity incubator for 24 hours, and the OD of each well of the cell plate was measured again. 600 Values ​​were recorded, and the OD values ​​before and after culture were calculated for each well. 600 The difference, i.e., ΔOD 600 This allows 0 ≤ OD before and after cultivation. 600 The lowest concentration of flavonoid components in a solution ≤0.05% can be determined as the minimum inhibitory concentration (MIC).

[0069] Using *Candida albicans* (CGMCC 2.4159) as the test strain (*Candida albicans* is a fungus used as an evaluation model), the ΔOD of flavonoid components in waste leaves of *Zizania latifolia* at different mass concentrations was investigated. 600 See Table 3 below for details.

[0070] Table 4. ΔOD of Candida albicans suspension at different mass concentrations of flavonoid components from discarded water chestnut leaves. 600

[0071]

[0072] The data in Table 4 show that when the concentration of flavonoids from waste water chestnut leaves is 1.25 mg / mL, the ΔOD of the Candida albicans suspension is... 600 When the ΔOD is 0.047 < 0.05 and the concentration is 0.625 mg / mL, 600 Since 0.054 > 0.05, it can be concluded that the MIC of the flavonoid component of waste leaves of water bamboo against Candida albicans CGMCC 2.4159 is 1.25 mg / mL.

[0073] Example 4: Preparation of a flavonoid antifungal spray from waste water chestnut leaves

[0074] Using the flavonoid components from waste leaves of water bamboo as the main ingredient, combined with ethanol, 1,2-propylene glycol, and polysorbate-80, a compound flavonoid spray is prepared through processes such as alcohol dissolution, miscibility, and filtration.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A preparation method of Zizania latifolia waste leaf flavonoid component, comprising the following steps: washing and drying fresh Zizania latifolia bracts to constant weight, crushing and sieving, and placing a certain amount of Zizania latifolia bract powder in a petroleum ether water bath to perform defatting by refluxing, to obtain defatted Zizania latifolia bract powder; adding the defatted Zizania latifolia bract powder to a certain amount of deionized water, and performing flash extraction by using a flash cell at a certain voltage for a certain time; after the end, adjusting the pH of the extraction liquid to 5.0±0.2, then adding a certain amount of cellulase, and performing enzymolysis for a certain time, then performing water bath enzyme inactivation, and then adding a certain amount of anhydrous ethanol and deionized water to adjust the final concentration of ethanol in the extraction liquid, and then performing extraction again, concentration, centrifugation, and freeze-drying to obtain a crude flavonoid component; the flash extraction voltage is 110 V, the flash extraction time is 4.1 min, the enzymolysis time is 60 min, and the enzyme dosage is 0.7%, and under the above conditions, the extraction rate of the Zizania latifolia waste leaf crude flavonoid component is 2.21±0.16%.

2. The preparation method according to claim 1, further comprising separating and purifying the prepared crude flavonoid component, and the specific method is: wetly packing a DM301 type macroporous resin which is activated, and using anhydrous acetone as an eluent, and then performing reduced pressure concentration and vacuum freeze-drying treatment on the effluent.

3. Use of the Zizania latifolia waste leaf flavonoid component prepared by the preparation method according to claim 2 in the preparation of an anti-Candida albicans product.

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