Method for extracting flavones from chrysanthemum morifolium ramat stem

Abstract

The present application belongs to the technical field of agricultural waste utilization, and particularly relates to a method for extracting flavones from chrysanthemum morifolium ramat straw. The method comprises the following steps: (1) cleaning, airing and crushing the chrysanthemum morifolium ramat straw to obtain chrysanthemum morifolium ramat straw powder; (2) inoculating the chrysanthemum morifolium ramat straw powder with a composite inoculant 1 to perform first-stage fermentation, and then inoculating the chrysanthemum morifolium ramat straw powder with a composite inoculant 2 to perform second-stage fermentation, thereby obtaining chrysanthemum morifolium ramat straw fermentation product; and (3) soaking the chrysanthemum morifolium ramat straw fermentation product in a deep eutectic solvent, then centrifuging to obtain supernatant, and filtering the supernatant through a microporous filter membrane, thereby obtaining chrysanthemum morifolium ramat straw flavone extract. The method can efficiently extract flavones from chrysanthemum morifolium ramat straw, improve the economic value of chrysanthemum morifolium ramat, and realize resource utilization of chrysanthemum morifolium ramat straw and turning waste into treasure.

Description

Methods for extracting flavonoids from Cymbidium goeringii stalks Technical Field

[0001] This invention belongs to the field of agricultural waste utilization technology, specifically relating to a method for extracting flavonoids from Chrysanthemum indicum straw. Background Technology

[0002] Chrysanthemum, a perennial herbaceous plant belonging to the Asteraceae family and the Chrysanthemum genus, is a commonly used traditional Chinese medicine and a plant designated by the Ministry of Health as both food and medicine, possessing significant medicinal and economic value. Traditional Chinese medicine theory holds that chrysanthemum is cool in nature, sweet and bitter in taste, and non-toxic, possessing the effects of dispelling wind and clearing heat, calming the liver and improving eyesight. Modern pharmacological studies have discovered that chrysanthemum contains various active ingredients such as volatile oils, chlorogenic acid, polysaccharides, and flavonoids, confirming that chrysanthemum has multiple biological functions, including anti-tumor, antiviral, antibacterial and anti-inflammatory effects, cardiovascular protection, and immune regulation. These studies provide a theoretical basis for the full utilization of chrysanthemum resources. Huibai chrysanthemum is a chrysanthemum variety with significant regional characteristics and excellent quality, widely cultivated in the Yellow River Delta region.

[0003] Currently, Huibai chrysanthemum is mainly used for processing its flowers into chrysanthemum tea or for direct medicinal use. After the flowers are harvested, the leaves, stems, and other stalks are mostly discarded. However, according to ancient Chinese medical texts, chrysanthemum stems and leaves have medicinal effects in treating sores, carbuncles, headaches, and dizziness, indicating that chrysanthemum stalks also contain similar active ingredients as chrysanthemum flowers. Modern analysis has shown that Huibai chrysanthemum stems, leaves, and stalks also contain total flavonoids, which have potential development and utilization value. The yield of Huibai chrysanthemum stalks is far higher than that of the flowers, and the waste of Huibai chrysanthemum stalks causes significant resource waste and environmental pollution. How to extract and recover the flavonoid components from Huibai chrysanthemum stalks and realize the resource utilization of Huibai chrysanthemum stalks is an important agricultural problem that urgently needs to be solved. Recycling and utilizing the flavonoid resources in Huibai chrysanthemum stalks and increasing the added value of Huibai chrysanthemum is of great significance for increasing income in the Huibai chrysanthemum industry and promoting rural revitalization. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for extracting flavonoids from Chrysanthemum indicum stalks. This method can efficiently extract flavonoids from Chrysanthemum indicum stalks, thereby increasing the economic value of Chrysanthemum indicum and realizing the resource utilization of Chrysanthemum indicum stalks, turning waste into treasure. The method of this invention is green, environmentally friendly, economical, efficient, and widely applicable.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for extracting flavonoids from Chrysanthemum indicum stalks includes the following steps:

[0007] (1) Wash, dry and crush the straw of Chrysanthemum indicum to obtain Chrysanthemum indicum straw powder;

[0008] (2) Inoculate compound microbial agent 1 into the powder of chrysanthemum straw for the first stage of fermentation, and then inoculate compound microbial agent 2 for the second stage of fermentation to obtain chrysanthemum straw fermentation product;

[0009] (3) The fermented product of Chrysanthemum indicum straw was soaked in a eutectic solvent, then centrifuged to obtain the supernatant. The supernatant was filtered through a microporous membrane to obtain Chrysanthemum indicum straw flavonoid extract.

[0010] Preferably, in step (1), the particle size of the Chrysanthemum morifolium straw powder is 150~200 mesh.

[0011] Preferably, in step (2), the compound microbial agent 1 is composed of Clostridium cellulose, Burkholderia brevis and Phanerochaete chrysosporium, with a mass ratio of 2:1:1, and the inoculation amount of the compound microbial agent 1 is 0.5% to 1.5% of the mass of Chrysanthemum stalk powder, preferably 1%.

[0012] Preferably, in step (2), the first stage of fermentation lasts for 2 to 3 days, the temperature is maintained at 40 to 45°C, preferably 45°C, the stirring rate is 10 to 20 r / min, the ventilation method is intermittent ventilation, ventilation for 5 to 10 minutes every 1 hour, and the ventilation rate is 0.02 to 0.05 L / kg dry weight / min.

[0013] Preferably, in step (2), the compound microbial agent 2 is composed of Vibrio fibrolyticus and Ruminococcus albus, with a mass ratio of 1:1, and the inoculation amount of the compound microbial agent 2 is 0.2~0.4% of the mass of Chrysanthemum stalk powder, preferably 0.4%.

[0014] Preferably, in step (2), the second stage fermentation lasts for 1 to 1.5 days, the temperature is maintained at 20 to 25°C, preferably 25°C, the stirring rate is 5 to 10 r / min, the ventilation method is continuous ventilation, and the ventilation rate is 0.2 to 0.3 L / kg dry weight / min.

[0015] Preferably, in step (2), nutrient solution is sprayed every 5-6 hours during the fermentation of Chrysanthemum morifolium straw powder. The spraying amount is 0.01-0.03 L / kg dry weight. The composition of the nutrient solution is: KH2PO4 20 mg / L, NH4Cl 40 mg / L, MgSO4·7H2O 30 mg / L, NiCl2·6H2O 5 mg / L, ZnSO4·7H2O 4 mg / L, H3BO4 0.05 mg / L, NiCl2·6H2O 0.08 mg / L, NaSeO4·10H2O 0.02 mg / L, and NaMoO4·2H2O 0.01 mg / L.

[0016] Preferably, in step (3), the eutectic solvent is composed of betaine, glucose and ethylene glycol in a molar ratio of 2:1:1 and has a water content of 20-30%.

[0017] Preferably, in step (3), the fermented chrysanthemum straw is soaked in a low eutectic solvent for 4-6 hours at a temperature of 50-80°C, preferably 80°C, and the solid-liquid ratio is 1:5-15 g / mL, preferably 1:10 g / mL.

[0018] Preferably, in step (3), the centrifugation speed is 10000~15000 r / min and the centrifugation time is 5~10 min.

[0019] Preferably, in step (3), the pore size of the microporous filter membrane is 0.1~0.15μm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The method described in this invention utilizes the straw of Chrysanthemum indicum, which was originally considered waste, to extract the medicinal component flavonoids, turning waste into treasure and increasing the added value and economic value of Chrysanthemum indicum.

[0022] 2. The method described in this invention extracts flavonoids through a two-step fermentation process, which is highly efficient and environmentally friendly. The extraction process avoids the use of traditional toxic and harmful solvents, and the conditions are mild, green, environmentally friendly, economical and efficient. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. 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.

[0024] It should be noted that the Huibai chrysanthemum straw used in the following embodiments all comes from Huimin County Jurun Agricultural Technology Co., Ltd. The Huibai chrysanthemum is a high-quality and high-yield chrysanthemum variety bred by the company through the exploration of medicinal chrysanthemum resources in the Yellow River Delta region. The standardized planting area in the Yellow River Delta has reached 20,000 mu, forming the largest chrysanthemum planting base north of the Yangtze River.

[0025] Example 1

[0026] The method for extracting flavonoids from Chrysanthemum indicum stalks according to the present invention includes the following steps:

[0027] (1) Wash and dry the chrysanthemum straw, then crush it to 150 mesh to obtain chrysanthemum straw powder.

[0028] (2) A compound microbial agent 1 consisting of Clostridium fibrinolyticum, Burkholderia brevis, and Phanerochaete chrysosporium in a mass ratio of 2:1:1 was inoculated into Chrysanthemum indicum straw powder at a mass ratio of 0.5% for the first stage of fermentation. The temperature was maintained at 40℃, the stirring rate was 10 r / min, and ventilation was carried out intermittently for 5 min every 1 h at a ventilation rate of 0.02 L / kg dry weight / min. The first stage of fermentation lasted for 2 days. Then, a compound microbial agent 2 consisting of Vibrio fibrinolyticus and Ruminococcus albus in a mass ratio of 1:1 was inoculated into the second stage of fermentation at a mass ratio of 0.2% for the second stage of fermentation. The temperature was maintained at 20℃, the stirring rate was 5 r / min, and the ventilation method was continuous ventilation at a ventilation rate of 0.2 L / kg dry weight / min. The second stage of fermentation lasted for 1 day. Nutrient solution was sprayed every 6 hours during the fermentation of Huibai chrysanthemum straw powder at a rate of 0.01 L / kg dry weight. The specific components of the nutrient solution were: KH₂PO₄ 20 mg / L, NH₄Cl 40 mg / L, MgSO₄·7H₂O 30 mg / L, NiCl₂·6H₂O 5 mg / L, ZnSO₄·7H₂O 4 mg / L, H₃BO₄ 0.05 mg / L, NiCl₂·6H₂O 0.08 mg / L, NaSeO₄·10H₂O 0.02 mg / L, and NaMoO₄·2H₂O 0.01 mg / L. After the first and second stages of fermentation, Huibai chrysanthemum straw powder was fermented to obtain Huibai chrysanthemum straw fermentate.

[0029] (3) The fermented product of Chrysanthemum morifolium straw was soaked in a eutectic solvent composed of betaine, glucose and ethylene glycol with a water content of 20% and a molar ratio of 2:1:1 for 4 hours at a temperature of 50°C and a solid-liquid ratio of 1:5 g / mL. Then, it was centrifuged at 10000 r / min for 10 min and the supernatant was collected. The supernatant was filtered through a microporous membrane with a pore size of 0.15 μm to obtain the flavonoid extract of Chrysanthemum morifolium straw.

[0030] Example 2

[0031] The method for extracting flavonoids from Chrysanthemum indicum stalks according to the present invention includes the following steps:

[0032] (1) Wash and dry the chrysanthemum straw, then crush it to 180 mesh to obtain chrysanthemum straw powder.

[0033] (2) A compound microbial agent 1, consisting of Clostridium fibrinolyticum, Burkholderia brevis, and Phanerochaete chrysosporium in a 2:1:1 ratio, was inoculated into Chrysanthemum morifolium straw powder at an inoculation rate of 1.0% for the first stage of fermentation. The temperature was maintained at 42℃, the stirring rate was 15 r / min, and ventilation was carried out intermittently at a rate of 0.03 L / kg dry weight / min every 1 hour for 8 minutes. The first stage of fermentation lasted for 3 days. Then, a compound microbial agent 2, consisting of Vibrio fibrinolyticus and Ruminococcus albus in a 1:1 ratio, was inoculated into the second stage of fermentation at an inoculation rate of 0.3%. The temperature was maintained at 23℃, the stirring rate was 8 r / min, and the ventilation method was continuous ventilation at a rate of 0.25 L / kg dry weight / min. The second stage of fermentation lasted for 1 day. Nutrient solution was sprayed every 6 hours during the fermentation of Huibai chrysanthemum straw powder at a rate of 0.02 L / kg dry weight. The specific components of the nutrient solution were: KH₂PO₄ 20 mg / L, NH₄Cl 40 mg / L, MgSO₄·7H₂O 30 mg / L, NiCl₂·6H₂O 5 mg / L, ZnSO₄·7H₂O 4 mg / L, H₃BO₄ 0.05 mg / L, NiCl₂·6H₂O 0.08 mg / L, NaSeO₄·10H₂O 0.02 mg / L, and NaMoO₄·2H₂O 0.01 mg / L. After the first and second stages of fermentation, Huibai chrysanthemum straw powder was fermented to obtain Huibai chrysanthemum straw fermentate.

[0034] (3) The fermented product of Chrysanthemum morifolium straw was soaked in a eutectic solvent composed of betaine, glucose and ethylene glycol with a water content of 25% and a molar ratio of 2:1:1 for 5 hours at a temperature of 70°C and a solid-liquid ratio of 1:10 g / mL. Then, it was centrifuged at 12000 r / min for 13 min and the supernatant was collected. The supernatant was filtered through a microporous membrane with a pore size of 0.12 μm to obtain the flavonoid extract of Chrysanthemum morifolium straw.

[0035] Example 3

[0036] The method for extracting flavonoids from Chrysanthemum indicum stalks according to the present invention includes the following steps:

[0037] (1) Wash and dry the chrysanthemum straw, then crush it to 200 mesh to obtain chrysanthemum straw powder.

[0038] (2) A compound microbial agent 1, consisting of Clostridium fibrinolyticum, Burkholderia brevis, and Phanerochaete chrysosporium in a 2:1:1 ratio, was inoculated into Chrysanthemum morifolium straw powder at a concentration of 1.5%. The first stage of fermentation was carried out at a temperature of 45℃, a stirring rate of 20 r / min, and intermittent ventilation at a rate of 0.05 L / kg dry weight / min for 10 min every 1 h. The first stage of fermentation lasted for 2 days. Then, a compound microbial agent 2, consisting of Vibrio fibrinolyticus and Ruminococcus albus in a 1:1 ratio, was inoculated into the second stage of fermentation at a concentration of 0.4%. The temperature was maintained at 25℃, the stirring rate was 10 r / min, and the ventilation method was continuous ventilation at a rate of 0.30 L / kg dry weight / min. The second stage of fermentation lasted for 1.5 days. Nutrient solution was sprayed every 6 hours during the fermentation of Huibai chrysanthemum straw powder at a rate of 0.03 L / kg dry weight. The specific components of the nutrient solution were: KH₂PO₄ 20 mg / L, NH₄Cl 40 mg / L, MgSO₄·7H₂O 30 mg / L, NiCl₂·6H₂O 5 mg / L, ZnSO₄·7H₂O 4 mg / L, H₃BO₄ 0.05 mg / L, NiCl₂·6H₂O 0.08 mg / L, NaSeO₄·10H₂O 0.02 mg / L, and NaMoO₄·2H₂O 0.01 mg / L. After the first and second stages of fermentation, Huibai chrysanthemum straw powder was fermented to obtain Huibai chrysanthemum straw fermented product.

[0039] (3) The fermented product of Chrysanthemum morifolium straw was soaked in a eutectic solvent composed of betaine, glucose and ethylene glycol with a water content of 30% and a molar ratio of 2:1:1 for 6 hours at 80℃ and a solid-liquid ratio of 1:15 g / mL. Then, it was centrifuged at 15000 r / min for 10 min and the supernatant was collected. The supernatant was filtered through a microporous membrane with a pore size of 0.1 μm to obtain the flavonoid extract of Chrysanthemum morifolium straw.

[0040] Comparative Example 1

[0041] The method for extracting flavonoids from Chrysanthemum indicum stalks is the same as in Example 1, except that a first-stage fermentation was not performed.

[0042] Comparative Example 2

[0043] The method for extracting flavonoids from Chrysanthemum indicum stalks is the same as in Example 1, except that a second stage of fermentation was not performed.

[0044] Comparative Example 3

[0045] The method for extracting flavonoids from Chrysanthemum indicum straw is the same as in Example 1, except that the inoculum used for the first stage of fermentation is Clostridium cellulose.

[0046] Comparative Example 4

[0047] The method for extracting flavonoids from Chrysanthemum indicum straw is the same as in Example 1, except that the inoculum used for the second stage of fermentation is Vibrio celluloseolyticus.

[0048] The flavonoid extracts from Chrysanthemum indicum straw obtained in Examples 1-3 and Comparative Examples 1-4 were tested, and the flavonoid extraction rate results are shown in Table 1.

[0049] Table 1 Flavonoid Extraction Rate

[0050]

Claims

1. A method for extracting flavonoids from Chrysanthemum indicum stalks, characterized in that, The process includes the following steps: (1) washing, drying, and crushing the straw of *Chrysanthemum indicum* to obtain *Chrysanthemum indicum* straw powder; (2) inoculating the *Chrysanthemum indicum* straw powder with compound microbial agent 1 for the first stage of fermentation, and then inoculating with compound microbial agent 2 for the second stage of fermentation to obtain *Chrysanthemum indicum* straw ferment; (3) soaking the *Chrysanthemum indicum* straw ferment in a eutectic solvent, then centrifuging to obtain the supernatant, and filtering the supernatant through a microporous membrane to obtain *Chrysanthemum indicum* straw flavonoid extract; in step (2), the compound microbial agent 1 is composed of *Clostridium cellulose*. The compound microbial agent 1 is composed of *Bacillus burgdorferi* and *Phanerochaete chrysosporium* in a mass ratio of 2:1:1, and the inoculation amount of compound microbial agent 1 is 0.5% to 1.5% of the mass of *Chrysanthemum indicum* straw powder; the compound microbial agent 2 is composed of *Vibrio cytolyticus* and *Ruminococcus flavonoids* in a mass ratio of 1:1, and the inoculation amount of compound microbial agent 2 is 0.2% to 0.4% of the mass of *Chrysanthemum indicum* straw powder; in step (3), the eutectic solvent is composed of betaine, glucose and ethylene glycol in a molar ratio of 2:1:1 and has a water content of 20% to 30%.

2. The method for extracting flavonoids from Chrysanthemum indicum stalks according to claim 1, characterized in that, In step (1), the particle size of the chrysanthemum straw powder is 150~200 mesh.

3. The method for extracting flavonoids from Chrysanthemum indicum stalks according to claim 1, characterized in that, In step (2), the first stage of fermentation lasts for 2-3 days, the temperature is maintained at 40-45℃, the stirring rate is 10-20 r / min, the ventilation method is intermittent ventilation, ventilation for 5-10 minutes every 1 hour, and the ventilation rate is 0.02-0.05 L / kg dry weight / min.

4. The method for extracting flavonoids from Chrysanthemum indicum stalks according to claim 1, characterized in that, In step (2), the second stage of fermentation lasts for 1 to 1.5 days, the temperature is maintained at 20 to 25°C, the stirring rate is 5 to 10 r / min, the ventilation method is continuous ventilation, and the ventilation rate is 0.2 to 0.3 L / kg dry weight / min.

5. The method for extracting flavonoids from Chrysanthemum indicum stalks according to claim 1, characterized in that, In step (2), nutrient solution is sprayed every 5-6 hours during the fermentation of chrysanthemum straw powder. The spraying amount is 0.01-0.03 L / kg dry weight. The composition of the nutrient solution is: KH2PO4 20 mg / L, NH4Cl 40 mg / L, MgSO4·7H2O 30 mg / L, NiCl2·6H2O 5 mg / L, ZnSO4·7H2O 4 mg / L, H3BO4 0.05 mg / L, NiCl2·6H2O 0.08 mg / L, NaSeO4·10H2O 0.02 mg / L, NaMoO4·2H2O 0.01 mg / L.

6. The method for extracting flavonoids from Chrysanthemum indicum straw according to claim 1, characterized in that, In step (3), the fermented chrysanthemum straw is soaked in a low eutectic solvent for 4-6 hours at a temperature of 50-80℃ and a solid-liquid ratio of 1:5-15 g / mL.

7. The method for extracting flavonoids from Chrysanthemum indicum stalks according to claim 1, characterized in that, In step (3), the centrifugation speed is 10000~15000 r / min, the centrifugation time is 5~10 min, and the pore size of the microporous filter membrane is 0.1~0.15 μm.

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