Method for extracting flavonoid compounds from lonicera caerulea leaves by microwave water bath method

Abstract

The invention belongs to the technical field of active substance extraction, and particularly relates to a method for extracting flavonoid compounds from lonicera caerulea leaves. The invention discloses a method for extracting flavonoid compounds from lonicera caerulea leaves by a microwave water bath method, which specifically comprises the following steps: drying and crushing fresh lonicera caerulea leaves to obtain lonicera caerulea leaf powder; adding an ethanol solution into the lonicera caerulea leaf powder, and carrying out microwave extraction and water bath to obtain a flavone crude extracting solution; and carrying out reduced pressure distillation and suction filtration, carrying out reduced pressure distillation on the obtained solution, and freeze-drying to obtain lonicera caerulea leaf flavonoid compound powder. According to the method, the flavonoid compounds are efficiently extracted from the lonicera caerulea leaves by utilizing an advanced extraction method and scientific experimental design.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance extraction, and particularly relates to a method for extracting flavonoids from Lonicera caerulea leaves. Background Art

[0002] Blue indigo fruit (Lonicera caerulea L.) is a deciduous shrub of the Caprifoliaceae family, commonly known as blue indigo honeysuckle, black blind fruit, goat milk fruit, and wild eggplant. It is primarily produced in the Russian Far East, North Korea, and Japan. In my country, it is primarily distributed in northeastern China, including Heilongjiang and Jilin, and also in Hebei, Shanxi, Ningxia, southern Gansu, Qinghai, northern Sichuan, and northwestern Yunnan. Due to its medicinal and edible properties, blue indigo fruit has broad application prospects, making it a high-quality berry with great market potential in cold regions.

[0003] Flavonoids are widely distributed in nature, primarily found in the leaves and fruits of plants. They are an important class of plant secondary metabolites. They possess a variety of active functions, including strong antioxidant capacity, which prevents oxidative damage by inhibiting and scavenging free radicals; cardiovascular effects, including blood pressure balance and platelet aggregation inhibition; and anti-tumor, anti-inflammatory, antibacterial, and hypoglycemic and lipid-lowering properties. Given these numerous active functions, flavonoids are increasingly being studied.

[0004] At present, the extraction of flavonoids often uses enzymatic hydrolysis, supercritical fluid carbon dioxide extraction, solvent extraction, ultrasonic extraction and microwave methods. Enzymatic hydrolysis has high enzyme cost, harsh reaction conditions, long reaction time and complex enzymatic hydrolysis products, and is difficult to operate; supercritical fluid carbon dioxide extraction has high equipment requirements, high cost and professional training, limited extraction range, large one-time investment, and product quality is affected by carbon dioxide purity; although the solvent extraction method is simple to operate, has low equipment requirements and low cost, it has problems such as long extraction time, high energy consumption and low economic benefits; although the ultrasonic extraction method has high extraction efficiency and good extraction quality, it has high equipment cost and complex maintenance, and the extraction effect is inconsistent after scale expansion, and may destroy certain components in the extract under certain conditions. The present invention uses microwave method combined with water bath extraction, which has the advantages of high yield, low energy consumption, simple operation, safety and environmental protection, flavonoid components are not easily destroyed and extraction is more complete. Summary of the Invention

[0005] The present invention provides a method for extracting flavonoids from blueberry leaves, which solves the technical gaps in the related art in extracting flavonoids from blueberry leaves.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for extracting flavonoids from Lonicera caerulea leaves comprises the following steps:

[0008] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0009] (2) Weigh 1 g of blue loquat leaf powder, add 50-80% ethanol solution at a solid-liquid ratio of 1 g / 30-60 mL, and soak for 60 min;

[0010] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 4 to 7 minutes, and the water bath heating temperature was 50 to 80° C.;

[0011] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0012] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0013] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0014] The present invention has the following beneficial effects:

[0015] Compared with the existing technology, the method for extracting flavonoids from blueberry leaves of the present invention has the advantages of simple operation, short time consumption and large extraction amount, has good practicality, and provides a reliable basis for extracting flavonoids from blueberry leaves.

[0016] The present invention combines microwave extraction with waterbath heating. Microwave extraction is performed five times, each lasting 4 to 7 minutes. Microwaves cause polar molecules within cells to rotate and rub at high speed, disrupting cell walls and promoting the rapid dissolution of flavonoids. Multiple extractions can improve the extraction rate. Subsequent waterbath heating further ensures complete extraction, while maintaining a temperature of 50 to 80°C to prevent damage to the structure and activity of flavonoids caused by excessive temperatures.

[0017] The present invention processes the ethanol extract through reduced pressure distillation, evaporating the ethanol at a lower temperature and reducing the possibility of flavonoids decomposing or oxidizing at high temperatures. Multiple volume adjustments, centrifugation, and secondary filtration steps effectively remove impurities, improve the purity of the extract, and ultimately yield a higher-quality powder of flavonoid compounds from the leaves of Lonicera edulis. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0019] Example 1

[0020] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0021] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0022] (2) Weigh 1 g of blue loquat leaf powder, add 50% ethanol solution at a solid-liquid ratio of 1 g / 60 mL, and soak for 60 min;

[0023] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 7 minutes, and the water bath heating temperature was 80°C;

[0024] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0025] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0026] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0027] Example 2

[0028] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0029] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0030] (2) Weigh 1 g of blue loquat leaf powder, add 60% ethanol solution at a solid-liquid ratio of 1 g / 40 mL, and soak for 60 min;

[0031] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 4 minutes, and the water bath heating temperature was 70°C;

[0032] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0033] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0034] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0035] Example 3

[0036] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0037] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0038] (2) Weigh 1 g of blue loquat leaf powder, add 70% ethanol solution at a solid-liquid ratio of 1 g / 50 mL, and soak for 60 min;

[0039] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 4 minutes, and the water bath heating temperature was 80°C;

[0040] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0041] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0042] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0043] Example 4

[0044] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0045] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0046] (2) Weigh 1 g of blue loquat leaf powder, add 70% ethanol solution at a solid-liquid ratio of 1 g / 60 mL, and soak for 60 min;

[0047] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 5 minutes, and the water bath heating temperature was 70°C;

[0048] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0049] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0050] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0051] Example 5

[0052] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0053] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0054] (2) Weigh 1 g of blue loquat leaf powder, add 50% ethanol solution at a solid-liquid ratio of 1 g / 50 mL, and soak for 60 min;

[0055] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 6 minutes, and the water bath heating temperature was 70°C;

[0056] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0057] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0058] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0059] Example 6

[0060] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0061] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0062] (2) Weigh 1 g of blue loquat leaf powder, add 60% ethanol solution at a solid-liquid ratio of 1 g / 60 mL, and soak for 60 min;

[0063] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 6 minutes, and the water bath heating temperature was 50°C;

[0064] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0065] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0066] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0067] Example 7

[0068] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0069] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0070] (2) Weigh 1 g of blue loquat leaf powder, add 70% ethanol solution at a solid-liquid ratio of 1 g / 30 mL, and soak for 60 min;

[0071] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 6 minutes, and the water bath heating temperature was 60°C;

[0072] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0073] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0074] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0075] Example 8

[0076] The method for extracting flavonoids from Lonicera caerulea leaves specifically comprises the following steps:

[0077] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0078] (2) Weigh 1 g of blue loquat leaf powder, add 50% ethanol solution at a solid-liquid ratio of 1 g / 40 mL, and soak for 60 min;

[0079] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 5 minutes, and the water bath heating temperature was 60°C;

[0080] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0081] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0082] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0083] Comparative Example 1

[0084] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0085] (2) Weigh 1 g of blue loquat leaf powder, add 60% ethanol solution at a solid-liquid ratio of 1 g / 50 mL, and soak for 60 min;

[0086] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 7 minutes, and the water bath heating temperature was 60°C;

[0087] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0088] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0089] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0090] Comparative Example 2

[0091] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0092] (2) Weigh 1 g of blue loquat leaf powder, add 80% ethanol solution at a solid-liquid ratio of 1 g / 60 mL, and soak for 60 min;

[0093] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 4 minutes, and the water bath heating temperature was 60°C;

[0094] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0095] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0096] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0097] Comparative Example 3

[0098] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0099] (2) Weigh 1 g of blue loquat leaf powder, add 50% ethanol solution at a solid-liquid ratio of 1 g / 30 mL, and soak for 60 min;

[0100] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 4 minutes, and the water bath heating temperature was 50°C;

[0101] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0102] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0103] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0104] Comparative Example 4

[0105] (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder;

[0106] (2) Weigh 1 g of blue loquat leaf powder, add 80% ethanol solution at a solid-liquid ratio of 1 g / 30 mL, and soak for 60 min;

[0107] (3) The mixed solution obtained in step (2) was subjected to microwave extraction for 5 times, and then heated in a water bath. The extract was collected and filtered with a filter membrane to obtain a filtrate; the microwave extraction time was 7 minutes, and the water bath heating temperature was 70°C;

[0108] (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml;

[0109] (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml;

[0110] (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a powder of flavonoid compounds from the leaves of Lonicera caerulea.

[0111] The content of total flavonoids in the extract of Lonicera caerulea leaves in the examples and comparative examples was determined.

[0112] Drawing of the standard curve: Accurately weigh 10 mg of the dried rutin standard, dissolve it with 60% ethanol solution by ultrasonication, and dilute it to 100 ml volumetric flask to prepare a rutin standard solution with a mass concentration of 100ug / ml. Accurately pipette 0.0 ml, 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml of the prepared rutin standard solution into a 10 ml volumetric flask. Add 0.3 ml of 5% NaNO2 and let it stand for 6 minutes; add 30.3 ml of 10% Al(NO3) and let it stand for 6 minutes; add 4.0 ml of 4% NaOH and let it stand for 15 minutes, and dilute it to 10 ml with 60% ethanol solution. Pour the completely reacted test solution into a 10 mm cuvette, use the blank solution as a reference, and perform UV-visible light scanning in the 600 nm to 400 nm band. Use the least squares method for linear regression to obtain the relationship between the rutin mass concentration ρ (μg / ml) and the absorbance A 505nm The linear regression equation of .

[0113] Flavonoid content determination method: Accurately pipette 1ml of the above extract into a 25ml volumetric flask for dilution and dilute with 60% ethanol. Pipette 5ml of the dilution into 5 10ml volumetric flasks in parallel as the test solution, measure the absorbance of each test solution, and take the average of the 5 parallel absorbance measurements as the A 505nm According to the linear regression equation, the absorbance A 505nm The mass concentration of the test solution ρ (μg / ml) can be obtained. The calculation formula is as follows: Total flavonoids content Where: ρ is the total flavonoids concentration (μg / ml); V1 is the fixed volume of the test solution, 10 ml; w is the multiple, 5 (25 ml diluent / 5 ml test solution); V2 is the total volume of the extract after fixed volume, 100 ml; 10 -3 It is the conversion multiple of μg / ml to mg / ml; m is the mass of the extracted material, 1g.

[0114] Table 1 Total flavonoids in the extracts from the leaves of Lonicera caerulea in the examples and comparative examples

[0115] From the data in Table 1, it can be seen that the contents of flavonoids in the extracts from the leaves of Indigofera caerulea in Examples 1-8 are higher than those in Comparative Examples 1-4, indicating that the technical solution of the present invention can increase the content of flavonoids in the extracts from the leaves of Indigofera caerulea.

[0116] Compared with Example 1, the flavonoid content in the blueberry leaf extract of Examples 5 and 8 is relatively low. This is because when extracting the blueberry leaf extract in Examples 5 and 8, the extraction time is shortened, the solid-liquid ratio is reduced, and the water bath temperature is reduced. This indicates that increasing the extraction time, solid-liquid ratio, and water bath temperature can increase the flavonoid content in the blueberry leaf extract.

[0117] Compared with Example 1, the content of flavonoids in the blue honeysuckle leaf extracts of Comparative Examples 2 and 4 was reduced. Increasing the ethanol concentration, reducing the extraction time, the solid-liquid ratio, and the water bath temperature did not increase the content of flavonoids in the blue honeysuckle leaf extract, indicating that too high an ethanol concentration is not conducive to the extraction of flavonoids in the blue honeysuckle leaf extract.

[0118] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting flavonoids from Lonicera caerulea leaves using a microwave water bath method, characterized in that: The following steps are involved: (1) Fresh blueberry leaves were dried at 60°C, ground and sieved through a 60-mesh sieve to obtain blueberry leaf powder; (2) Weigh 1 g of blue loquat leaf powder, add 50-80% ethanol solution, and soak for 60 min; (3) The mixed solution obtained in step (2) was subjected to microwave extraction 5 times, heated in a water bath, and the extract was collected and filtered using a filter membrane to obtain a filtrate; (4) distilling the ethanol extract obtained in step (3) under reduced pressure to a volume of 100 ml; (5) The solution obtained in step (4) was centrifuged and filtered twice and the volume was adjusted to 100 ml; (6) The solution obtained in step (5) is distilled under reduced pressure into a paste, and then freeze-dried to obtain a crude extract powder of Lonicera caerulea leaves. In step (2), the solid-liquid ratio of the Lonicera edulis leaf powder to the ethanol solution is 1 g / 30-60 mL; The microwave extraction time in step (3) is 4 to 7 minutes, and the water bath heating temperature is 50 to 80°C.

2. The method for extracting flavonoids from Lonicera caerulea leaves according to claim 1, wherein: The Lonicera caerulea leaf powder is obtained by the following method: The blue honeysuckle leaves are washed, dried and crushed to obtain blue honeysuckle leaf powder, wherein the blue honeysuckle leaf powder has a particle size of 0.5-1 cm.

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