Amomum villosum flavonoid compound with antibacterial and anti-inflammatory effects as well as preparation method and application of amomum villosum flavonoid compound

Abstract

The invention relates to an antibacterial and anti-inflammatory amomum villosum flavone compound as well as a preparation method and application thereof. The method comprises the following steps: uniformly mixing a natural deep eutectic solvent and water to obtain a natural deep eutectic solvent aqueous solution; adding the Amomum villosum powder into the natural deep eutectic solvent aqueous solution, uniformly mixing, extracting the Amomum villosum powder by using an ultrasonic-assisted technology, and centrifugally filtering after the extraction to obtain an Amomum villosum flavone extracting solution; adsorbing and eluting the flavone extracting solution of the amomum villosum on a column, and enriching and recovering the flavonoid compounds in the extracting solution. According to the method, the amomum villosum is extracted through ultrasonic-assisted NaDES, the macroporous resin enrichment technology is combined, efficient extraction of the flavonoid compounds of the amomum villosum and sustainable utilization of the natural deep eutectic solvent are achieved, and the flavonoid compounds obtained through enrichment have anti-inflammatory activity, antibacterial activity and the like; the problems that in the prior art, the extraction yield of the flavonoids of the amomum villosum is not high, and environment friendliness is not achieved are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and preparation, and particularly relates to an Amomum villosum flavonoid compound with antibacterial and anti-inflammatory effects, a preparation method and application thereof. Background Art

[0002] Amomum villosum Lour. (A. villosum) is a plant of the genus Cardamom in the ginger family. Its dried mature fruit has a long history of medicinal use as a traditional Chinese medicine in China. Amomum villosum is mainly distributed in Guangdong, Guangxi, Yunnan and other provinces. The authentic production area is Yangchun City, Guangdong Province, and the largest production area is Yunnan Province. Amomum villosum is widely used in traditional Chinese medicine because of its unique medicinal value of removing dampness and appetizing, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. The pharmacologically active ingredients of Amomum villosum are the key factors for its pharmacological effects. Studies have reported that Amomum villosum contains a variety of active ingredients such as flavonoids, volatile oils, polysaccharides, polyphenols, and organic acids. Among them, flavonoids, as plant-specific secondary metabolites, have attracted much attention due to their antioxidant, anti-inflammatory, and anti-tumor biological activities. However, the extraction efficiency of flavonoids in Amomum villosum is currently low, and the research on pharmacological effects is still to be deepened.

[0003] According to existing literature research, the method for extracting Amomum villosum flavonoids is generally organic solvent extraction, which is often assisted by heating reflux, ultrasound, microwave and other technologies to improve the yield. However, there are many disadvantages, such as large amount of organic solvent used, long extraction time, low extraction efficiency, high energy consumption, high cost, and potential harm to the environment and human health.

[0004] Therefore, developing a new method for extracting flavonoids from Amomum villosum to improve the extraction efficiency and yield is of great significance for the in-depth research and wide application of Amomum villosum. Summary of the invention

[0005] In order to solve the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing flavonoid compounds of Amomum villosum with antibacterial and anti-inflammatory properties. The method extracts Amomum villosum by ultrasound-assisted NaDES, and combines macroporous resin enrichment technology to achieve efficient extraction of Amomum villosum flavonoid compounds and sustainable utilization of natural deep eutectic solvents, and the enriched flavonoid compounds have anti-inflammatory and antibacterial activities, solving the problems of low extraction rate of Amomum villosum flavonoid compounds and environmental unfriendliness in the prior art.

[0006] Another object of the present invention is to provide a Amomum villosum flavonoid compound prepared by the above method.

[0007] Another object of the present invention is to provide the application of the Amomum villosum flavonoids compound.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing a Amomum villosum flavonoid compound, comprising the following steps:

[0010] (1) mixing a natural deep eutectic solvent and water to obtain a natural deep eutectic solvent aqueous solution; then adding Amomum villosum powder to the natural deep eutectic solvent aqueous solution and mixing them evenly, extracting the Amomum villosum powder using ultrasound-assisted technology, and centrifuging and filtering after the extraction to obtain an Amomum villosum flavonoid extract; wherein the natural deep eutectic solvent hydrogen bond acceptor is betaine or proline, and the hydrogen bond donor is glycerol, propylene glycol, urea, lactic acid, malic acid, citric acid, fructose, xylitol, glucose or sorbitol;

[0011] (2) The Amomum villosum flavonoids extract obtained in step (1) is subjected to column adsorption and elution to enrich and recover the flavonoid compounds in the extract, and the natural deep eutectic solvent is recovered and reused.

[0012] Preferably, the Amomum villosum powder in step (1) is obtained by crushing the Amomum villosum raw material and passing it through a 40-mesh sieve.

[0013] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the natural deep eutectic solvent in step (1) is 1:(1-4).

[0014] Preferably, the natural deep eutectic solvent in step (1) is betaine-glycerol.

[0015] Preferably, the method for preparing the natural deep eutectic solvent in step (1) is as follows: the hydrogen bond donor and the hydrogen bond acceptor are mixed in proportion, and the mixture is heated at 80-100° C. under stirring until a transparent and uniform liquid is formed; the mixed natural deep eutectic solvent is placed in a vacuum drying oven for vacuum drying for 24 hours to remove moisture; finally, the prepared natural deep eutectic solvent is stored in a desiccator before use, and is placed for one month without precipitation of crystals, thereby obtaining a stable, uniform and transparent natural deep eutectic solvent.

[0016] Preferably, the water content of the natural deep eutectic solvent aqueous solution in step (1) is 10-50% (w / w), most preferably 40%.

[0017] Preferably, the solid-liquid ratio of the Amomum villosum powder to the natural deep eutectic solvent aqueous solution in step (1) is 1:(10-50) g / mL, most preferably 1:20 g / mL.

[0018] Preferably, the power of the ultrasonic extraction in step (1) is 60-100%P, most preferably 80%P.

[0019] Preferably, the temperature of the ultrasonic extraction in step (1) is 40-80°C, most preferably 70°C.

[0020] Preferably, the ultrasonic extraction time in step (1) is 10-90 minutes, most preferably 30 minutes.

[0021] Preferably, the centrifugation frequency in step (1) is 3000-5000 rpm, and the centrifugation time is 10-20 minutes.

[0022] Preferably, the specific steps of step (2) include:

[0023] A1. Pretreatment of macroporous resin: Soak the macroporous resin (AB-8 type) in anhydrous ethanol for 24 hours to allow it to fully swell before wet column loading. Wash the column with 3-5 times the volume of deionized water until there is no ethanol smell.

[0024] A2. Recovery of Amomum villosum flavonoids: The Amomum villosum flavonoids extract was loaded onto the column at a flow rate of 30-40 mL / h, the water-soluble NaDES was washed off with water at the same flow rate, and then eluted with 80-90% (v / v) ethanol at 30-40 mL / h, the eluate was collected, and the ethanol and water were removed by a vacuum rotary evaporator, and the Amomum villosum flavonoids were enriched and recovered;

[0025] A3. Recovery of natural deep eutectic solvent: Use a vacuum rotary evaporator to remove water from the NaDES washed with water, and the recovered NaDES can be reused to extract Amomum villosum flavonoids, thereby completing the recovery of the natural deep eutectic solvent.

[0026] The Amomum villosum flavonoids prepared by the above method have good antibacterial and anti-inflammatory effects, and can be used to prepare antibacterial drugs, anti-inflammatory drugs, health products or cosmetics. Therefore, the Amomum villosum flavonoids prepared by the above method and the application of the Amomum villosum flavonoids in anti-inflammatory and antibacterial should all be within the protection scope of the present invention.

[0027] Key points of the technical solution of the present invention:

[0028] 1. Selection of natural deep eutectic solvent (NaDES): The present invention uses natural deep eutectic solvent composed of plant metabolic components as the extraction medium, which is not only safe and low-toxic, but also can maintain uniformity and stability during the extraction process, effectively avoiding solvent precipitation and crystallization. NaDESs and flavonoids in Amomum villosum interact through hydrogen bonds, significantly improving the dissolution rate and extraction efficiency of flavonoids.

[0029] 2. Ultrasonic-assisted extraction technology: The present invention utilizes the "cavitation effect" of ultrasonic technology to effectively destroy the cell wall of Amomum villosum, allowing NaDESs to penetrate deeper into the cells, thereby enhancing the extraction effect of flavonoid compounds. Compared with traditional organic solvent extraction methods, ultrasonic-assisted extraction has the advantages of simple operation, time saving, environmental protection and safety.

[0030] 3. Macroporous resin enrichment and recovery technology: During the extraction process, the present invention uses macroporous resin adsorption technology to effectively separate the Amomum villosum flavonoids from NaDESs, while realizing the recovery and reuse of NaDESs. This step not only simplifies the purification process of the extract, improves the purification efficiency, but also reduces the production cost, laying a foundation for the industrial production of Amomum villosum flavonoids.

[0031] The technical solution advantages of the present invention are:

[0032] 1. High efficiency: Through the combination of NaDESs and ultrasound-assisted technology, the present invention significantly improves the extraction efficiency of flavonoid compounds from Amomum villosum, with a higher yield and shorter extraction time.

[0033] 2. Environmental protection: NaDESs are made of natural ingredients, are safe, low-toxic, and recyclable, reducing environmental pollution and waste of resources. At the same time, ultrasound-assisted technology is more environmentally friendly than traditional organic solvent extraction methods.

[0034] 3. Economical: The introduction of macroporous resin enrichment and recovery technology not only simplifies the purification process of the extract, but also reduces production costs and improves economic benefits.

[0035] 4. Industrialization potential: The preparation method of Amomum villosum flavonoid compounds provided by the present invention is simple, efficient, environmentally friendly, and easy to realize industrial production, and has broad market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the standard curve of rutin.

[0037] Figure 2 The results are a comparison of the flavonoid extraction rates obtained by using NaDES aqueous solution with different water contents during the extraction of flavonoid compounds from Amomum villosum.

[0038] Figure 3 This is a comparison of the flavonoid extraction rates obtained by using different solid-liquid ratios of NaDES aqueous solution and Amomum villosum powder during the extraction of flavonoid compounds from Amomum villosum.

[0039] Figure 4 This is a comparison of the flavonoid extraction rates obtained under different ultrasonic powers during the extraction of flavonoid compounds from Amomum villosum.

[0040] Figure 5 This is a comparison of the flavonoid extraction rates obtained under different ultrasonic extraction temperatures during the extraction of flavonoid compounds from Amomum villosum.

[0041] Figure 6 This is a comparison of the flavonoid extraction rates obtained under different ultrasonic extraction times during the extraction of flavonoid compounds from Amomum villosum.

[0042] Figure 7 This is a diagram of the thermal properties of the natural deep eutectic solvent synthesized in Example 1.

[0043] Figure 8 These are the morphology pictures of Amomum villosum powder samples after extraction with different solvents.

[0044] Fig. 9 The effects of different concentrations of Amomum villosum flavonoids on the viability of RAW264.7 cells (a) and their effects on inhibiting the production of NO in RAW264.7 cells (b). DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional practices, methods and equipment in the art. The reagents and materials used in the present invention are all commercially available.

[0046] Example 1 Preparation of Amomum villosum flavonoids extract

[0047] (1) Betaine and glycerol are placed in a stoppered conical flask at a molar ratio of 1:2, and the mixture is heated at 85°C under magnetic stirring until a transparent and uniform liquid is formed. The prepared NaDES is placed in a vacuum drying oven and vacuum dried for 24 hours to remove moisture. If no crystals are precipitated at room temperature for one month, it is a natural deep eutectic solvent;

[0048] (2) mixing the natural deep eutectic solution and deionized water to obtain a NaDES aqueous solution with a water content of 30% (w / w);

[0049] (3) Grind the sand into powder using a crusher, pass through a 40-mesh sieve, and store at room temperature;

[0050] (4) Mix the Amomum villosum powder and the NaDES aqueous solution at a solid-liquid ratio of 1:30 g / mL, place the centrifuge tube in an ultrasonic instrument, ultrasonic power 85% P (ultrasonic power 100% P = 360w), ultrasonic temperature 50°C, and extraction time 30 minutes;

[0051] (5) centrifuging the mixed solution to obtain the upper layer of Amomum villosum flavonoids extract; the centrifugal speed is 5000 r / min, and the centrifugal time is 5 minutes;

[0052] (6) The flavonoid content was determined by aluminum nitrate colorimetry, and the total flavonoid extraction rate was calculated.

[0053] Example 2-30

[0054] The specific steps refer to Example 1, which differs from Example 1 in that the natural deep eutectic solvent, water content, extraction temperature, extraction time, material-liquid ratio, ultrasonic power, etc. are different. See the parameters listed in Table 1 for details.

[0055] Comparative Example 1 Preparation of Amomum villosum ethanol extract

[0056] Refer to Example 25, except that the betaine-glycerol aqueous solution is replaced with an ethanol solution, and the other conditions are the same as in Example 25.

[0057] Comparative Example 2 Preparation of Amomum villosum methanol extract

[0058] Refer to Example 25, except that the betaine-glycerol aqueous solution is replaced with a methanol solution, and the other conditions are the same as in Example 25.

[0059] Comparative Example 3 Preparation of Amomum villosum water extract

[0060] Refer to Example 25, except that the betaine-glycerol aqueous solution is replaced with deionized water, and the other conditions are the same as in Example 25.

[0061] Table 1 Parameter settings of Examples 1-30 and Comparative Examples 1-3

[0062]

[0063]

[0064] Example 31 A method for recovering Amomum villosum flavonoid compounds

[0065] (1) Pretreatment of macroporous resin: Weigh 10 g of AB-8 macroporous resin and soak it in anhydrous ethanol for 24 h to allow it to fully swell before wet column loading. Wash the column with 5 volumes of deionized water until there is no ethanol smell.

[0066] (2) Recovery of Amomum villosum flavonoid compounds: The Amomum villosum flavonoid extract of Example 25 was loaded onto the column at a flow rate of 30-40 mL / h, and the water-soluble NaDES was washed off with deionized water at the same flow rate, and then eluted with 80-90% (v / v) ethanol at 30-40 mL / h. The eluate was collected, and the ethanol and water were removed using a vacuum rotary evaporator. The Amomum villosum flavonoid compounds were enriched and recovered, and the content of flavonoid compounds in the extract and the recovery rate were calculated.

[0067] Example 32 A method for recovering and reusing a natural deep eutectic solvent

[0068] (1) collecting the portion washed with deionized water in Example 31, evaporating the water using a vacuum rotary evaporator, and reusing the portion to extract Amomum villosum flavonoids;

[0069] (2) The recovered natural deep eutectic solvent with a water content of 30% was used to extract Amomum villosum flavonoids again according to the extraction steps and conditions of Example 25 above to obtain a crude extract, and the flavonoid content was detected and compared with that of the first extraction.

[0070] Test Example 1 Determination of flavonoid content

[0071] (1) Rutin standard preparation: Accurately weigh an appropriate amount of rutin reference substance, add ethanol to prepare a reference substance mother solution with a mass concentration of 1 mg / mL, mix well, and store in a 4°C refrigerator for later use. Take an appropriate amount of rutin reference substance mother solution and dilute it with ethanol to obtain standard solutions with concentrations of 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively. Take 200 μL of rutin standard solution of different concentrations, place them in 1.5 mL centrifuge tubes, add 40 μL of 5% sodium nitrite aqueous solution, mix well, and let stand for 6 minutes; then add 40 μL of 10% aluminum nitrate aqueous solution, mix well, and let stand for 6 minutes; finally, add 400 μL of 1 mol / L sodium hydroxide solution and 400 μL of deionized water, mix well, and let stand for 15 minutes; take the supernatant and measure the absorbance value at a wavelength of 513 nm. Plot the rutin reference substance concentration (mg / mL) as the horizontal axis and the absorbance as the vertical axis, and calculate the regression equation and correlation coefficient, such as Figure 1 shown.

[0072] Step (1) The linear regression equation is: y = 0.7743x + 0.1, R 2 =0.9988. The results showed that the rutin reference solution showed a good linear relationship in the concentration range of 0.1-0.7 mg / ml.

[0073] (2) Determination of flavonoid content

[0074] The Amomum villosum flavonoids extract obtained in Examples 1-30 and Comparative Examples 1-3 was diluted 5 times with ethanol, 200 μL was taken and placed in a 1.5 mL centrifuge tube, 40 μL of 5% sodium nitrite aqueous solution was added in sequence, mixed, and allowed to stand for 6 min; 40 μL of 10% aluminum nitrate aqueous solution was added, mixed, and allowed to stand for 6 min; finally, 400 μL of 1 mol / L sodium hydroxide solution and 400 μL of deionized water were added, mixed, and allowed to stand for 15 min; the supernatant was taken and the absorbance value was measured at a wavelength of 513 nm. The flavonoid content in the Amomum villosum flavonoids extract obtained in Examples 1-30 and Comparative Examples 1-3 was determined according to the standard curve, and the extraction rate of Amomum villosum flavonoids was calculated according to the formula "extraction rate (%) = (flavonoid concentration × dilution multiple × extract volume) / Amomum villosum powder mass × 100%".

[0075] (3) Content determination results: The results are shown in Table 2.

[0076] Table 2 The results of the determination of the flavonoids extraction rate of Amomum villosum

[0077]

[0078] As can be seen from Table 2, the highest extraction rate of Amomum villosum flavonoids in Examples 1-10 is the extraction with betaine glycerol in Example 1, and combined with the stability of NaDES, betaine-glycerol (molar ratio of 1:2) was used as the optimal solvent for subsequent single factor experiment optimization of extraction conditions.

[0079] Examples 1 and 11-30 are examples of the present invention that use single factor optimization to sequentially optimize the relevant extraction factors such as water content, material-liquid ratio, ultrasonic power, extraction temperature, extraction time, etc., to ensure that the flavonoids extracted by each factor are under the best conditions, and then optimize the next factor, so that the extraction rate of Amomum villosum flavonoids is significantly improved, thereby significantly improving the utilization rate of Amomum villosum raw materials. The optimization results of the water content factor in Examples 1 and 11-14 are as follows: Figure 2 As shown, Figure 2 The horizontal axis is the water content (w / w), and the vertical axis is the extraction rate of Amomum villosum flavonoids. Because a notable feature of NaDES is that it has a certain viscosity, it is difficult to mix it with the powder, which affects the extraction rate of active compounds. Adding an appropriate amount of water to NaDES can significantly reduce its concentration and increase its polarity. However, if excessive water is added, the hydrogen bond interaction of NaDES will be weakened or even disappear, thereby affecting the extraction effect. Figure 2 It can be seen that when the water content of NaDES is 20-40% (w / w), the extraction rate of flavonoid components is relatively high. Taking the cost factor into consideration, betaine glycerol with a water content of 40% (w / w) was initially selected as the extraction solvent, and the subsequent extraction process was optimized.

[0080] The optimization results of the liquid-to-solid ratio factors of Examples 1 and 15-18 are as follows Figure 3 As shown in the figure, the horizontal axis is the liquid-to-solid ratio, and the vertical axis is the extraction rate of Amomum villosum flavonoids. It can be seen from the figure that the extraction rate of Amomum villosum flavonoids is the highest at 1: (20-50) g / mL. Increasing the amount of extraction solvent can make the solvent fully contact the sample material, which is conducive to the mass transfer of active substances. However, too high a liquid-to-solid ratio will cause solvent waste and increase the extraction cost. Therefore, the initial solid-liquid ratio is 1:20 g / mL, and the subsequent extraction process optimization is continued.

[0081] The results of ultrasonic power factor optimization in Examples 16, 19-22 are as follows Figure 4 As shown in the figure, the horizontal axis is the ultrasonic power, and the vertical axis is the extraction rate of Amomum villosum flavonoids. It can be seen from the figure that the extraction rate of Amomum villosum flavonoids is the highest at 80%P. The greater the ultrasonic power, the more intense the collapse process of the cavitation bubble, and the larger the cavitation bubble will be, which will generate greater shear force and promote the extraction process. On the other hand, higher ultrasonic power will lead to a greater sonochemical effect during the cavitation bubble rupture process, thereby promoting the degradation of flavonoids.

[0082] The results of the optimization of the extraction temperature factor of Examples 22-26 are as follows Figure 5 As shown in the figure, the horizontal axis is the extraction temperature, and the vertical axis is the extraction rate of Amomum villosum flavonoids. It can be seen from the figure that the extraction rate of Amomum villosum flavonoids is the highest at 70-80℃. This may be because the increase in temperature enhances the cavitation effect during ultrasonic extraction, accelerates the movement of solvent molecules, and reduces the viscosity of the natural deep eutectic solvent, so that more and more target compounds penetrate deep from the inside.

[0083] The results of optimizing the extraction time factor in Examples 25, 27-30 are as follows Figure 6 As shown in the figure, the horizontal axis is the extraction time, and the vertical axis is the extraction rate of Amomum villosum flavonoids. It can be seen from the figure that the extraction rate of Amomum villosum flavonoids is the highest at 30 minutes, and the extraction rate increases with the increase of extraction time. This may be due to the fact that at this stage the solvent dissolves surface compounds and intracellular components through diffusion and osmosis processes respectively, and the ultrasound assistance will lead to the aggravation of cell wall rupture of plant materials, reducing the restriction of cell structure on mass transfer process. The reason for the decrease in the extraction rate of Amomum villosum flavonoids in the later period may be that the ultrasound time is too long, resulting in the degradation of flavonoids.

[0084] Compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3, the extraction rate of Amomum villosum flavonoids in ethanol, methanol, and water extracts extracted under the same conditions is 2.2-3.6 times higher than that in Example 25. Therefore, betaine-glycerol (molar ratio 1:2) is selected as the optimal solvent.

[0085] Test Example 2 Natural Deep Eutectic Solvothermal Analysis

[0086] The natural deep eutectic solvent synthesized in Example 1 was thermally analyzed using a synchronous thermal analyzer. About 20 mg of the sample was placed in a 70 μL aluminum crucible and heated from 30°C to 400°C at a rate of 10°C / min under the protection of nitrogen.

[0087] Thermal properties such as Figure 7 shown.

[0088] As can be seen from the figure, the solvolysis temperature of the natural deep eutectics synthesized from betaine glycerol is higher than 70°C, which indicates that they can remain stable within 70°C to extract flavonoids from Amomum villosum.

[0089] Test Example 3 Microstructure of Amomum villosum powder after extraction with different solvents

[0090] Scanning electron microscopy (SEM) was used to analyze the degree of surface damage of Amomum villosum powder after extracting Amomum villosum flavonoids with betaine glycerol (30% water content, molar ratio 1:2), ethanol, methanol and water. The instrument used was Czech TESCAN MIRALMS. The specific steps are as follows:

[0091] (1) Sample washing: The powders extracted from Example 25, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were washed alternately with ethanol and water for 3 times;

[0092] (2) Sample freeze-drying: The washed powder sample was pre-frozen at -80°C for 12 h, and then placed in a freeze dryer for 24 h;

[0093] (3) SEM analysis: A small amount of powder was directly adhered to the conductive adhesive and gold was sprayed for 45 seconds using a Quorum SC7620 sputtering coater at 10 mA. The sample morphology was then photographed using a TESCAN MIRALMS scanning electron microscope.

[0094] Test results such as Figure 8 shown.

[0095] As shown in the figure, the surface of the original powder is irregular and blocky, and the surface is smooth and flat. The surface structure of the powder treated with the natural deep eutectic solvent is destroyed, indicating that the method of the present invention has a stronger dissolution effect on the target and is more conducive to the mass transfer process. The powder obtained by ethanol, methanol and water maintains a relatively complete structure.

[0096] Test Example 4 Recovery rate of Amomum villosum flavonoids and solution and extraction efficiency of the recovered solvent for the next extraction of flavonoids

[0097] (1) The recovery rate of Amomum villosum flavonoids was calculated according to the formula "recovery rate of Amomum villosum flavonoids (%) = flavonoid content of supernatant after recovery / flavonoid content of extract × 100%".

[0098] (2) The solution recovery rate was calculated according to the formula “solution recovery rate (%) = amount of recovered solution (mL) / sample amount (mL) × 100%”.

[0099] (3) The extraction efficiency was calculated according to the formula "extraction efficiency (%) = flavonoid content of the second extraction solution / flavonoid content of the first extraction solution × 100%".

[0100] The flavonoid content test in Test Example 4 is consistent with the flavonoid content detection method of Test Example 1 using the aluminum nitrate colorimetric method.

[0101] The test results are shown in Table 3.

[0102] Table 3 Amomum villosum flavonoids recovery rate, solution recovery rate and extraction efficiency of the recovered solvent

[0103]

[0104] As shown in the table, the recovery rate of Amomum villosum flavonoids is 82.61%. The NaDES used in the present invention is water-soluble and can be eluted with water. The volume of the recovered pure NaDES is 19 mL, and the solution recovery rate is 90.48%. Amomum villosum flavonoids are extracted again according to the method of Example 25, and the extraction rate of flavonoid compounds is 7.67%, and the extraction efficiency is 94.71%, which still has a good extraction rate, preliminarily proving the recyclability and reuse ability of NaDES.

[0105] Test Example 5 Antibacterial Effect of Flavonoids from Amomum villosum

[0106] The serial dilution method was used to determine the inhibitory effects on pathogenic bacteria Staphylococcus aureus and conditional pathogenic bacteria Propionibacterium acnes and their MIC and MBC. Staphylococcus aureus (ATCC 25923) and Propionibacterium acnes (ATCC 11827) were both from Guangdong Provincial Microbiological Culture Collection Center.

[0107] (1) Bacterial transfer

[0108] Take out the bacterial culture from the cryopreservation tube, pick up a small amount with a culture loop, and streak it on a plate. When colonies grow, this plate is named F1. Pick out a pure single colony from the F1 medium and streak it again. When colonies grow in the medium, this plate is F2. F1 plates can be stored at 4°C for one month, while F2 plates can only be stored at 4°C for one week.

[0109] (2) Preparation of bacterial suspension

[0110] Take several single colonies on the F2 plate, suspend the colonies in saline, and adjust the turbidity to OD600 = 0.2 by adding more bacteria or saline to reach 10 8CFU / mL. Dilute the bacterial saline solution 100 times with CAMHB or BHI medium to prepare the bacterial stock solution.

[0111] (3) Determination of MIC of Amomum villosum flavonoids by two-fold dilution method

[0112] A1. Sample dilution: add 4 mg / mL sample (flavonoids of Amomum villosum) into the first well of a U-shaped 96-well plate, 50 μL per well, and dilute from the second well to the tenth well. The 11th well is a bacterial solution as a blank control, and the 12th well is a culture medium without sample and bacterial solution as a negative control;

[0113] A2, MIC: Add 50 μL of bacterial solution to each well, and the final bacterial concentration in each well is 5×10 5 CFU / mL, seal the 96-well plate with sealing film, incubate for 16-20h or 48h, and observe the results. The lowest sample concentration at which the growth of microorganisms in the wells is completely inhibited by naked eyes is the MIC;

[0114] A3, MBC: Determine the minimum bactericidal concentration by smearing the plate. Take 50 μL of the supernatant in the clear well and dilute it in a gradient manner. Use an L-shaped diffuser to evenly smear it on the plate, incubate it overnight, and observe the results. The minimum sample concentration that does not grow bacteria on the plate is the MBC. The test results are shown in Table 4.

[0115] Table 4

[0116]

[0117] It can be seen that the higher the concentration of the prepared Amomum villosum flavonoids, the better the inhibitory effect on the strain, especially for the conditional pathogen Propionibacterium acnes, the minimum bactericidal concentration is 500 μg / mL, indicating that the Amomum villosum flavonoids prepared by the present invention are suitable for preparing antibacterial drugs or anti-acne cosmetics.

[0118] Test Example 6 Anti-inflammatory Effect of Flavonoids from Amomum villosum

[0119] (1) Cell recovery

[0120] Remove cells from cryopreservation tubes and thaw them quickly in a 37°C water bath. Wipe cryopreservation tubes with 75% alcohol and place them in a clean bench. Use a pipette to aspirate the cell suspension in the cryopreservation tubes and drop it into a 15mL centrifuge tube containing 7mL complete culture medium. Centrifuge at 1000rpm for 3min, remove the upper culture medium, add 2mL culture medium to resuspend the cells, transfer the cell solution to a culture dish containing 8mL culture medium, use the inverted "8" method to gently shake the culture dish to evenly distribute the cells, and place it in a 37°C, 5% CO 2 The cells were cultured in an incubator and the cell survival and status were observed on the next day.

[0121] (2) Cell passaging

[0122] When the cells reached the logarithmic growth phase, the old culture medium was removed, and 2 mL of PBS was added to rinse once to remove floating cells. 4 mL of fresh culture medium was added to gently blow the cells off the bottom of the dish, collect them in a 15 mL centrifuge tube, and gently blow them evenly. After mixing, 0.5 mL of cell solution was transferred to a culture dish containing 8 mL of culture medium. The culture dish was gently shaken using the inverted "8" method to evenly distribute the cells, and then placed in a 37°C, 5% CO 2 Culture in an incubator.

[0123] (3) Cell cryopreservation

[0124] For cells blown off during cell passaging, discard the upper layer of culture medium, add 1 mL of freezing solution, seal the freezing tube and freeze the cells using the gradient cooling method. The gradient cooling program is: 4°C, 30 min; -20°C, 30 min; -80°C, overnight, and finally store in a liquid nitrogen tank.

[0125] (4) Cell counting and plating

[0126] Take 100 μL of the mixed cell suspension and add it to a centrifuge tube containing 900 μL of complete culture medium. Gently blow and take 10 μL of the diluted cell solution and add it to the upper and lower blocks of the counting plate respectively. Count under a microscope. The counting principle is "count the upper part but not the lower part, count the left but not the right part". Count the number of cells in the four squares of the upper and lower areas. Divide the total number of cells in the two areas by 2 to get the average number of cells in the four squares of a single area, which is recorded as N. The cell solution density is N / 4×10×10 4 Pieces / mL.

[0127] (5) CCK8 assay to measure cell viability

[0128] The CCK8 method was used to measure the effect of Amomum villosum flavonoids on the viability of RAW264.7 cells. The cell fluid was counted and diluted to 5.0×10 4 Each well of the 96-well plate was inoculated with 100 μL of cell dilution buffer and placed in a 37°C, 5% CO 2 Culture in an incubator. After 24 hours of culture, add 100 μL of complete cell culture medium containing the sample. After another 24 hours of culture, remove the sample and culture medium. Add 100 μL of culture medium containing 10% CCK8 reagent to each well in the dark. Continue to culture for 3 hours. Use an enzyme reader to measure the absorbance at 450 nm. The formula for calculating cell survival rate is as follows:

[0129] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0130] As: absorbance of experimental wells (containing cells, culture medium, CCK8 reagent and drug solution)

[0131] Ac: Absorbance of control well (containing cells, culture medium, CCK8 reagent, but no drug)

[0132] Ab: absorbance of blank well (containing culture medium and CCK8 reagent, but not cells or drugs)

[0133] (6) Determination of NO release

[0134] The Bio-Tech NO Kit was used to measure the effect of Amomum villosum flavonoids on the inhibition of NO production in RAW264.7 cells. The cell fluid was counted and diluted to 5.0×10 5 Each well of the 96-well plate was inoculated with 100 μL of cell dilution buffer and placed in a 37°C, 5% CO 2 Cultured in an incubator, after 24 hours of culture, 50 μL of sample solution and 50 μL of LPS solution were added, and the final concentration of the LPS group was 1 μg / mL; the same volume of cell basal culture medium was the blank group. After 24 hours of co-culture, 50 μL of supernatant was aspirated, and the NO release in the cell supernatant was detected using a NO kit. The OD value at 540 nm was recorded using a multifunctional microplate reader. The effect of total flavonoids on NO release was expressed as the OD ratio of the treatment group to the blank group. The experiment was repeated 3 times. The test results are as follows. Fig. 9 shown.

[0135] The anti-inflammatory activity of Amomum villosum flavonoids was evaluated by detecting the NO release rate induced by LPS in RAW264.7 cells. Fig. 9 a in the figure is the viability of RAW264.7 cells. Fig. 9 b in is the NO release rate. Fig. 9 As known from a in the figure, 100, 200, and 400 μg / mL of Amomum villosum flavonoids had no significant effect on the viability of RAW264.7 cells and had no significant toxicity. Fig. 9 As shown in b, the NO release rate of the LPS group was higher than that of the blank group, indicating that the inflammatory model was successfully established. The NO release rates of the Amomum villosum flavonoid compounds at different concentrations were lower than those of the LPS group and were concentration-dependent, indicating that the Amomum villosum flavonoid compounds prepared by the present invention have excellent anti-inflammatory effects and are suitable for the preparation of anti-inflammatory drugs or soothing cosmetics.

[0136] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing flavonoid compounds from Amomum villosum, characterized in that: The following steps are involved: (1) mixing a natural deep eutectic solvent and water to obtain a natural deep eutectic solvent aqueous solution; then adding Amomum villosum powder to the natural deep eutectic solvent aqueous solution and mixing them evenly, then extracting the Amomum villosum powder using ultrasound-assisted technology, and centrifugally filtering after the extraction to obtain an Amomum villosum flavonoid extract; The hydrogen bond acceptor of the natural deep eutectic solvent is betaine or proline, and the hydrogen bond donor is glycerol, propylene glycol, urea, lactic acid, malic acid, citric acid, fructose, xylitol, glucose or sorbitol; (2) subjecting the Amomum villosum flavonoids extract obtained in step (1) to column adsorption and elution to enrich and recover the flavonoid compounds in the extract.

2. The method according to claim 1, characterized in that The Amomum villosum powder in step (1) is obtained by crushing the Amomum villosum raw material and passing it through a 40-mesh sieve.

3. The method according to claim 1, characterized in that The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor of the natural deep eutectic solvent in step (1) is 1:(1-4).

4. The method according to claim 1, characterized in that: The natural deep eutectic solvent in step (1) is betaine-glycerol.

5. The method according to claim 1, characterized in that The water content of the natural deep eutectic solvent aqueous solution in step (1) is 10-50% (w / w).

6. The method according to claim 1, characterized in that The solid-liquid ratio of the Amomum villosum powder to the natural deep eutectic solvent aqueous solution in step (1) is 1:(10-50) g / mL.

7. The method according to claim 1, characterized in that The power of the ultrasonic extraction in step (1) is 60-100%P, the temperature of the ultrasonic extraction is 40-80°C, and the time of the ultrasonic extraction is 10-90 minutes; The centrifugal frequency in step (1) is 3000-5000 rpm, and the centrifugal time is 10-20 minutes.

8. The method according to claim 1, characterized in that The specific steps of step (2) include: A1. Soak the macroporous resin in anhydrous ethanol for 24 hours to allow it to fully swell and then wet pack the column. Wash the column with 3-5 times the volume of deionized water until there is no ethanol smell. A2, loading the Amomum villosum flavonoids extract onto the column at a flow rate of 30-40 mL / h, washing off the water-soluble natural deep eutectic solvent with water at the same flow rate, and then eluting with 80-90% (v / v) ethanol at 30-40 mL / h, collecting the eluate, removing ethanol and water using a vacuum rotary evaporator, and enriching and recovering the Amomum villosum flavonoids; A3. Using a vacuum rotary evaporator, water in the natural deep eutectic solvent washed with water is removed to recover the natural deep eutectic solvent.

9. A flavonoid compound of Amomum villosum prepared by the method according to any one of claims 1 to 8.

10. Use of the Amomum villosum flavonoids compound according to claim 9 in the preparation of antibacterial drugs, anti-inflammatory drugs or cosmetics.

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