A method for extracting total flavones from bamboo leaves

Abstract

The application provides an extraction method of total flavones of bamboo leaves, and relates to the technical field of plant flavone extraction. The extraction method is a variable-temperature cycle drying treatment of low-temperature freezing and hot air drying of bamboo leaves, and combines with subsequent subcritical water combined with high-voltage pulse electric field treatment to effectively extract flavone components in the bamboo leaves. The total flavone content of the extract is optimized through subsequent ultrasonic alcohol extraction and acid precipitation, so that the total flavone content of the final extract after drying is more than 50%, the extraction effect of the total flavones of bamboo leaves is comprehensively improved, and a good foundation is further provided for subsequent purification of the extract.

Description

Technical Field

[0001] This invention relates to the field of plant flavonoid extraction technology, specifically to a method for extracting total flavonoids from bamboo leaves. Background Technology

[0002] Flavonoids in bamboo leaves have broad application prospects in the food, medicine, and daily chemical industries due to their various biological activities, including anti-oxidation, antibacterial, blood lipid regulation, and cardiovascular protection. They are also widely used in food additives, pharmaceutical intermediates, functional foods, and cosmetics.

[0003] my country is rich in bamboo resources, and extracting total flavonoids from bamboo leaves has significant economic benefits. Currently, extraction methods for total flavonoids from bamboo leaves mainly fall into four categories: chemical extraction, biological extraction, physical extraction, and combined extraction. Chemical extraction is the most traditional method, including water extraction and organic solvent extraction. Water extraction is economical and environmentally friendly but has a low extraction rate and produces many impurities. Organic solvent extraction has a relatively high extraction rate but suffers from significant solvent loss and pollution. Biological extraction mainly involves enzymatic hydrolysis, which, while improving the extraction rate, requires precise control of the hydrolysis conditions. Physical extraction mainly involves high temperature, high pressure, and microwave conditions, but the overall extraction efficiency for bamboo leaves is not high. Combined extraction is currently a research hotspot, combining multiple technologies to leverage synergistic advantages. However, the final total yield of total flavonoids from bamboo leaves is still relatively low, and the total flavonoid content in the extract is often low, posing challenges for subsequent fine processing. Therefore, it is crucial to provide a method that can effectively improve the extraction rate of total flavonoids from bamboo leaves and optimize the total flavonoid content in the extracted product. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting total flavonoids from bamboo leaves, which effectively improves the extraction rate of total flavonoids from bamboo leaves and further increases the content of total flavonoids in the extract, facilitating subsequent purification.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for extracting total flavonoids from bamboo leaves, the extraction method comprising the following steps:

[0007] S1. Bamboo leaf pretreatment: Place the bamboo leaves in a hot air environment of -10-15℃ and 35-45℃ for 4-5 cycles, with a total cycle time of 20-28 hours, to obtain dried bamboo leaves for later use.

[0008] S2. After pulverizing the above-mentioned dried bamboo leaves, add them to warm water at 60-80℃, keep them in a water bath for 30-50 minutes, then extract them with subcritical water combined with a high-voltage pulse electric field for 20-30 minutes, and then cool them to room temperature to obtain the preliminary extract for later use.

[0009] S3. Add ethanol to the preliminary extract and adjust the ethanol concentration to 50%-60%. Then, sonicate for 15-20 minutes to obtain the treated material for later use.

[0010] S4. After removing ethanol, adjust the system to acidity, perform acid precipitation, centrifuge to remove the supernatant, wash the precipitate with ice water until neutral, and then dry to obtain the total flavonoid extract of bamboo leaves.

[0011] Preferably, in step S1, the treatment time at -10-15°C is 1-2 hours, and the treatment time in a hot air environment at 35-45°C is 4-5 hours.

[0012] Preferably, in step S2, the ratio of dried bamboo leaves pulverized and then added to warm water at 60-80℃ is 1:12-18.

[0013] Preferably, the specific method for subcritical water combined with high-voltage pulsed electric field extraction in step S2 is to adjust the system pressure to 0.5-1MPa, the temperature to 120-130℃, and to introduce a high-voltage electric field into the system, controlling the electric field strength to 18-22kV / cm and the number of pulses to 15-20.

[0014] Preferably, the power of the ultrasonic treatment in step S3 is 300-500W.

[0015] Preferably, the ethanol removal method in step S4 is to remove ethanol at a water bath temperature of 70-80°C.

[0016] Preferably, the specific method for adjusting the system to be acidic in step S4 is to use HCl to adjust the pH of the system to 3.5-4.5.

[0017] Preferably, the acid precipitation treatment in step S4 is carried out at a temperature of 60-65°C for 4-8 hours.

[0018] Preferably, the drying method in step S4 is constant temperature drying at 40-50℃.

[0019] This invention provides a method for increasing the total flavonoid content in extracts in one step, which has the following advantages compared to existing technologies:

[0020] This invention employs variable-temperature cyclic drying of bamboo leaves, combined with subsequent subcritical water treatment using a high-voltage pulsed electric field to effectively extract flavonoids from the leaves. Subsequent ultrasonic alcohol extraction and acid precipitation optimize the total flavonoid content of the extract, resulting in a final dried extract with a total flavonoid content exceeding 50%. This comprehensively improves the extraction efficiency of total flavonoids from bamboo leaves and provides a solid foundation for subsequent extract purification. Attached Figure Description

[0021] Figure 1This is the rutin reflectance absorption curve of the present invention;

[0022] Figure 2 This is the standard curve of rutin in this invention;

[0023] Figure 3 This is a schematic diagram comparing the extraction rates of total flavonoid extracts from bamboo leaves in the embodiments and comparative examples of the present invention;

[0024] Figure 4 This is a schematic diagram comparing the flavonoid content in the total flavonoid extract of bamboo leaves in the embodiments and comparative examples of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1:

[0027] Extraction of total flavonoids from bamboo leaves:

[0028] (1) Place the bamboo leaves in a -10°C freezer for 2 hours, then place them in a 30°C hot air environment for 5 hours. Repeat the above operation 4 times to obtain dried bamboo leaves. Then pulverize the dried bamboo leaves and pass them through a 60-mesh sieve to obtain bamboo leaf powder.

[0029] (2) Add bamboo leaf powder to 70°C warm water at a material-to-liquid ratio of 1:12, keep it in a water bath for 40 minutes, then raise the temperature to 120°C and pressurize it to 1 MPa. Maintain the subcritical water environment for 30 minutes, and during this period, introduce a high-voltage electric field with an electric field strength of 18 kV / cm into the system. At the same time, set the pulse number to 18. After the subcritical water treatment is completed, allow it to cool naturally to room temperature to obtain the preliminary extract.

[0030] (3) Add anhydrous ethanol to the preliminary extract, adjust the ethanol concentration to 55%, and then treat it with ultrasound at 400W power for 15 minutes to obtain the treated material for later use.

[0031] (4) The above-mentioned material was subjected to ethanol removal at a water bath temperature of 75°C. After removing the ethanol, HCl was added to the system to adjust the pH of the system to 4. The system was subjected to acid precipitation at 60°C for 6 hours. After centrifugation to remove the supernatant, the precipitate was washed with ice water until neutral and then dried at a constant temperature of 45°C to obtain the total flavonoid extract of bamboo leaves.

[0032] Example 2:

[0033] Extraction of total flavonoids from bamboo leaves:

[0034] (1) Place the bamboo leaves in a -15°C freezer for 1 hour, then place them in a 45°C hot air environment for 4 hours. Repeat the above operation 5 times to obtain dried bamboo leaves, then pulverize them and pass them through a 60-mesh sieve to obtain bamboo leaf powder.

[0035] (2) Add bamboo leaf powder to 70℃ warm water at a material-to-liquid ratio of 1:15, keep it in a water bath for 40 minutes, then raise the temperature to 130℃ and the pressure to 0.8 MPa, maintain the subcritical water environment for 20 minutes, and during this period, pass a high-voltage electric field with an electric field strength of 22 kV / cm into the system, and set the pulse number to 15. After the subcritical water treatment is completed, let it cool naturally to room temperature to obtain the preliminary extract.

[0036] (3) Add anhydrous ethanol to the preliminary extract, adjust the ethanol concentration to 50%, and then treat it with ultrasonic power of 300W for 20 minutes to obtain the treated material for later use.

[0037] (4) The above-mentioned material was subjected to ethanol removal at a water bath temperature of 75°C. After removing the ethanol, HCl was added to the system to adjust the pH of the system to 4. The system was subjected to acid precipitation at 60°C for 6 hours. After centrifugation to remove the supernatant, the precipitate was washed with ice water until neutral and then dried at a constant temperature of 45°C to obtain the total flavonoid extract of bamboo leaves.

[0038] Comparative Example 1:

[0039] Extraction of total flavonoids from bamboo leaves:

[0040] The extraction method of Comparative Example 1 is basically the same as that of Example 2, except for step (1), which is as follows:

[0041] (1) The bamboo leaves were frozen at -15°C for 5 hours and then placed in a hot air environment at 45°C for 20 hours to obtain dried bamboo leaves. The dried bamboo leaves were then crushed and passed through a 60-mesh sieve to obtain bamboo leaf powder.

[0042] Comparative Example 2:

[0043] Extraction of total flavonoids from bamboo leaves:

[0044] The extraction method of Comparative Example 2 is basically the same as that of Example 2, except for step (1), which is as follows:

[0045] (1) Bamboo leaves are placed in a hot air environment at 45℃ for 20 hours to obtain dried bamboo leaves, which are then crushed and passed through a 60-mesh sieve to obtain bamboo leaf powder.

[0046] Comparative Example 3:

[0047] Extraction of total flavonoids from bamboo leaves:

[0048] The extraction method of Comparative Example 3 is basically the same as that of Example 2, except for step (2), which is as follows:

[0049] (2) Add bamboo leaf powder to 70℃ warm water at a material-to-liquid ratio of 1:15, keep warm in water bath for 40 minutes, then raise the temperature to 130℃, raise the pressure to 0.8Mpa, maintain the subcritical water environment for 20 minutes, then cool naturally to room temperature and then pass through a high voltage electric field with an electric field strength of 22kV / cm, while setting the pulse number to 15 to obtain the preliminary extract;

[0050] Comparative Example 4:

[0051] Extraction of total flavonoids from bamboo leaves:

[0052] The extraction method of Comparative Example 4 is basically the same as that of Example 2, except for step (2), which is as follows:

[0053] (2) Add bamboo leaf powder to 70℃ warm water at a material-to-liquid ratio of 1:15, keep warm in water bath for 40 minutes, then pass through a high voltage electric field with an electric field strength of 22kV / cm, and set the pulse number to 15 to obtain preliminary extract;

[0054] Comparative Example 5:

[0055] Extraction of total flavonoids from bamboo leaves:

[0056] The extraction method of Comparative Example 5 is basically the same as that of Example 2, except for step (2), which is as follows:

[0057] (2) Add bamboo leaf powder to 70℃ warm water at a material-to-liquid ratio of 1:15, keep warm in a water bath for 40 minutes, then raise the temperature to 130℃, raise the pressure to 0.8Mpa, maintain the subcritical water environment for 20 minutes, and then cool naturally to room temperature to obtain the preliminary extract.

[0058] Comparative Example 6:

[0059] Extraction of total flavonoids from bamboo leaves:

[0060] The extraction method of Comparative Example 6 is basically the same as that of Example 2, except for step (2), which is as follows:

[0061] (2) Add bamboo leaf powder to 70℃ warm water at a material-to-liquid ratio of 1:15, keep warm in a water bath for 40 minutes, and then cool naturally to room temperature to obtain preliminary extract;

[0062] Detection:

[0063] Total flavonoid extracts of bamboo leaves were obtained by extracting them from bamboo leaves harvested at the end of November in the same batch in autumn using the methods described in the above examples and comparative examples.

[0064] 1. The extraction rate of total flavonoids from bamboo leaves and the flavonoid content in the total flavonoids from bamboo leaves were determined in each group;

[0065] (1) The extraction rate was detected as follows: The dried and pulverized bamboo leaf powder of each group was weighed and recorded as m1. Then the total flavonoid extract of bamboo leaves obtained in each group was weighed and recorded as m2. The extraction rate of the total flavonoid extract of bamboo leaves was calculated as follows:

[0066] Extraction rate % = m2 / m1 × 100%;

[0067] (2) The method for detecting flavonoid content is as follows:

[0068] The content of flavonoids in the total flavonoid extract of bamboo leaves was determined by the NaNO2-Al(NO3)3 colorimetric method.

[0069] Standard curve plotting

[0070] Accurately weigh 10 mg of rutin standard into a 100 mL volumetric flask, dissolve and dilute with 50% ethanol to obtain a 0.1 mg / mL rutin standard solution.

[0071] Accurately measure 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of rutin standard solution respectively, transfer them to 10 mL test tubes, add 50% ethanol to a final volume of 2.0 mL, add 0.5 mL of 50 g / L NaNO2 solution, mix well, and let stand for 5 min. Add 0.5 mL of 100 g / L Al(NO3)3 solution, mix well, and let stand for 5 min. Add 2.0 mL of 40 g / L NaOH solution, mix well, and let stand for 15 min. (The maximum absorption wavelength of the rutin colorimetric reaction solution in the visible light region (400~800 nm) is 509 nm. (The rutin reflectance absorption curve is shown in Figure 1). Figure 1 (As shown). Therefore, the detection wavelength for the colorimetric reaction of bamboo leaf flavonoid samples was set at 509 nm, and the absorbance value was measured at 509 nm.

[0072] Plot a standard curve for rutin with absorbance as the x-axis and concentration as the y-axis (e.g., Figure 2 As shown in the figure, the linear equation derived from the standard curve of rutin is y = 189.39x + 0.3048, with a coefficient of determination R0. 2 The value is 1.0000, indicating a good linear fit, which can be used to determine the flavonoid content in the total flavonoid extract of bamboo leaves.

[0073] Weigh out M (0.1 g) of the total flavonoid extract samples from each group, dissolve them in 50% ethanol solution, and make up to a final volume of V (100 mL). For the determination, take the supernatant for analysis (the supernatant is diluted by N, N=2). Substitute the measured absorbance into the standard curve to calculate the concentration C (y value, mg / mL) of bamboo leaf flavonoids. The flavonoid content in the total flavonoid extract of bamboo leaves is then calculated as follows:

[0074] Flavonoid content % = (C×V×N) / (M×1000)×100%.

[0075] The specific test results are shown in Table 1 below:

[0076] Table 1

[0077] Group Extraction rate (%) Concentration (mg / mL) Flavonoid content (%) Example 1 2.011 0.2621 52.42 Example 2 2.024 0.2765 55.30 Comparative Example 1 1.874 0.2458 49.16 Comparative Example 2 1.821 0.2216 44.32 Comparative Example 3 1.937 0.2105 42.10 Comparative Example 4 1.472 0.1479 29.58 Comparative Example 5 1.231 0.1018 20.36 Comparative Example 6 0.975 0.0971 19.42

[0078] As can be seen from the above tests, the methods of Example 1 and Example 2 can effectively improve the extraction rate of total flavonoids from bamboo leaves and increase the content of flavonoids in the extract. Compared with Comparative Example 4 and Comparative Example 5, the combination of subcritical water and pulsed electric field in Example 2 can significantly improve the final extraction effect compared with the use of single conditions.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting total flavonoids from bamboo leaves, characterized in that, The extraction method includes the following steps: S1. Bamboo leaf pretreatment: Place the bamboo leaves in a hot air environment of -10-15℃ and 35-45℃ for 4-5 cycles, with a total cycle time of 20-28 hours, to obtain dried bamboo leaves for later use. S2. After pulverizing the above-mentioned dried bamboo leaves, add them to warm water at 60-80℃, keep them in a water bath for 30-50 minutes, then extract them with subcritical water combined with a high-voltage pulse electric field for 20-30 minutes, and then cool them to room temperature to obtain the preliminary extract for later use. S3. Add ethanol to the preliminary extract and adjust the ethanol concentration to 50%-60%. Then, sonicate for 15-20 minutes to obtain the treated material for later use. S4. After removing ethanol, adjust the system to acidity, perform acid precipitation, centrifuge to remove the supernatant, wash the precipitate with ice water until neutral, and then dry to obtain the total flavonoid extract of bamboo leaves.

2. The extraction method according to claim 1, characterized in that: In step S1, the treatment time at -10 to -15°C is 1 to 2 hours, and the treatment time in a hot air environment at 35 to 45°C is 4 to 5 hours.

3. The extraction method according to claim 1, characterized in that: In step S2, the ratio of dried bamboo leaves pulverized and then added to warm water at 60-80℃ is 1:12-18.

4. The extraction method according to claim 1, characterized in that: The specific method for subcritical water combined with high-voltage pulsed electric field extraction in step S2 is to adjust the system pressure to 0.5-1MPa, the temperature to 120-130℃, and to introduce a high-voltage electric field into the system, controlling the electric field strength to 18-22kV / cm and the number of pulses to 15-20.

5. The extraction method according to claim 1, characterized in that: The power of the ultrasonic treatment in step S3 is 300-500W.

6. The extraction method according to claim 1, characterized in that: In step S4, ethanol is removed by performing ethanol removal at a water bath temperature of 70-80℃.

7. The extraction method according to claim 1, characterized in that: The specific method for adjusting the system to be acidic in step S4 is to use HCl to adjust the pH of the system to 3.5-4.

5.

8. The extraction method according to claim 1, characterized in that: In step S4, the acid precipitation treatment is carried out at a temperature of 60-65℃ for 4-8 hours.

9. The extraction method according to claim 1, characterized in that: The drying method in step S4 is constant temperature drying at 40-50℃.

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