A gongyin powder extract, a preparation method and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a radix herbae erigerontis extract, a preparation method and application thereof.The present application provides a preparation method of radix herbae erigerontis extract, comprising the following steps: (1) after the radix herbae erigerontis is crushed, the radix herbae erigerontis is mixed with ethanol for immersion, ultrasonic extraction, centrifugal separation to obtain an alcohol extract and filter residue; (2) the filter residue is mixed with water, and subcritical extraction is carried out to obtain a filtrate; (3) the alcohol extract and the filtrate are respectively concentrated and dried, and then combined to obtain the radix herbae erigerontis extract.The radix herbae erigerontis extract is successfully prepared by using ultrasonic wave auxiliary + subcritical extraction method, the extract has good antibacterial property on S.aureus, E.coli and S.agalactiae, and has good in-vitro antioxidant activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a dandelion extract, its preparation method, and its application. Background Technology

[0002] Bovine mastitis is one of the most common diseases in dairy farming. The main symptoms include redness, swelling, heat, and pain in the udder, as well as fever and loss of appetite in affected cows. In severe cases, it can lead to death. Bovine mastitis causes significant economic losses to dairy farming, including reduced milk production, decreased milk quality, and increased treatment costs. Furthermore, it leads to increased culling rates and decreased reproductive performance. Therefore, active prevention and treatment of bovine mastitis in clinical practice is of great importance for ensuring the health of dairy cows, improving farming efficiency, and guaranteeing the quality of dairy products.

[0003] Microbial infection is one of the main causes of mastitis in dairy cows. Currently, the most common treatment is the use of antibiotics, but this leads to problems such as bacterial resistance and antibiotic residues, increasing the difficulty of treatment. Therefore, there is an urgent clinical need for a green, safe, and effective drug formulation to replace antibiotics in the treatment of mastitis in dairy cows.

[0004] Currently, traditional Chinese medicine (TCM) shows promising application prospects in the treatment of mastitis in dairy cows. It can effectively alleviate clinical symptoms such as redness, swelling, heat, and pain in the mammary glands, promote blood circulation in dairy cows, accelerate inflammation resolution, and has advantages such as low toxicity and cost. Dandelion powder is a classic formula for treating mastitis in the second part of the Chinese Veterinary Pharmacopoeia, possessing the effects of clearing heat and detoxifying, reducing swelling and dispersing carbuncles. Clinically, dandelion powder is often mixed with feed. Affected cows often exhibit symptoms such as lethargy, loss of appetite, or even complete anorexia, making it difficult to accurately control the feed intake of each cow during feeding management. This significantly reduces the therapeutic effect of dandelion powder on mastitis in dairy cows. In addition, there are problems such as the first-pass effect of drugs after passing through the gastrointestinal tract and liver, blocked milk ducts affecting drug diffusion, and the poor absorption of many insoluble components in TCM. Therefore, changing the dosage form and administration route of dandelion powder is beneficial for its better efficacy in clinical practice. Summary of the Invention

[0005] The purpose of this invention is to provide a dandelion extract, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing dandelion extract, comprising the following steps:

[0008] (1) After crushing the dandelion powder, mix it with ethanol and soak it. Extract it by ultrasonication, centrifuge it to obtain the supernatant and filter residue.

[0009] (2) The filter residue was mixed with water and then subjected to subcritical extraction to obtain the filtrate;

[0010] (3) The alcohol extract and the filtrate were concentrated and dried separately and then combined to obtain Dandelion extract;

[0011] In step (1), the temperature of the ultrasound is set to 30-90℃; the power of the ultrasound is set to 350-450W; and the mass-to-volume ratio of the dandelion powder to ethanol is 1g:20-40mL.

[0012] The mass-to-volume ratio of the filter residue to water in step (2) is 1g:12-16mL; the subcritical extraction temperature is 120-210℃ and the pressure is 2-4MPa.

[0013] Preferably, the volume fraction of ethanol in step (1) is 80-90%; and the soaking time is 25-35 min.

[0014] Preferably, the ultrasound time in step (1) is set to 50-70 min;

[0015] Preferably, the centrifugation speed in step (1) is 2000-3000 rpm; the centrifugation time is 8-12 min.

[0016] Preferably, the pH of the water in step (2) is 8.2 to 10.2; and the subcritical extraction time is 30 to 70 min.

[0017] The present invention also provides the application of the dandelion extract in the preparation of an antibacterial agent for bovine mastitis.

[0018] Preferably, the pathogen causing the bovine mastitis is one or more of Staphylococcus aureus, Escherichia coli, or Streptococcus agalactiae.

[0019] The present invention also provides the application of the dandelion extract in the preparation of antioxidants.

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

[0021] This invention successfully prepared a dandelion extract using ultrasound-assisted subcritical extraction. The extract exhibits good antibacterial activity against S. aureus, E. coli, and S. agalactiae, and also demonstrates good in vitro antioxidant activity. Attached Figure Description

[0022] Figure 1 This is the standard curve for chlorogenic acid in Example 2;

[0023] Figure 2 Example 2: Effects of different factors on the yield of chlorogenic acid from dandelion extract;

[0024] Figure 3 The response surface diagram of the interaction between two factors in the dandelion extract of Example 2 is shown.

[0025] Figure 4 The appearance of the EGP lyophilized powder in Example 2;

[0026] Figure 5 The Fourier transform infrared absorption spectrum of the dandelion extract in Example 3 is shown. Detailed Implementation

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Test materials

[0029] Table 1 Main Reagents

[0030]

[0031] Table 2 Main Instruments and Equipment

[0032]

[0033]

[0034] Example 1

[0035] A method for preparing dandelion extract (EGP) includes the following steps:

[0036] The dandelion powder (60g dandelion, 60g honeysuckle, 60g forsythia, 30g loofah sponge, 25g tetrapanax papyriferus, 25g hibiscus leaves, 30g Fritillaria thunbergii) was ground to 80 mesh using a grinder. The powder was then soaked in 85% ethanol for 30 minutes at a liquid-to-solid ratio of 25:1 (v / w). Following this, ultrasonic extraction was performed at 35°C and 430W for 60 minutes. After extraction, the mixture was cooled to room temperature, centrifuged for 10 minutes, and the supernatant was collected and freeze-dried under vacuum. The supernatant was then stored at -20°C.

[0037] Example 2

[0038] 1. Determination of Dandelion Extraction Rate

[0039] 1.1 Construction of the chlorogenic acid standard curve

[0040] Dandelion extract contains a high amount of chlorogenic acid; therefore, its yield is used as an indicator to determine the extraction efficiency. Chlorogenic acid standard was dissolved in 60% methanol solution to obtain a standard stock solution of 20.00 μg / mL. The stock solution was then diluted with 60% methanol solution to obtain standard solutions with mass concentrations of 2.00, 4.00, 6.00, 8.00, 10.00, 12.00, 14.00, and 16.00 μg / mL. The absorbance at 329 nm was measured, and a standard curve was plotted. Figure 1 Within the chlorogenic acid concentration range of 2–16 μg / mL, a good linear relationship was observed, with the linear regression equation being Y = 0.0475X + 0.0015, R0. 2 =0.9995.

[0041] 1.2 Calculation of Dandelion Extraction Rate

[0042] The absorbance of the ethanol extract of dandelion obtained under different extraction conditions was measured at 329 nm, and the yield of chlorogenic acid was calculated according to formula (1).

[0043]

[0044] In the formula: Y represents the yield of chlorogenic acid; N is the dilution factor; V is the volume of the test liquid in the cuvette; W is the weight of one dose of Dandelion Powder.

[0045] 1.3 Preparation and optimization results of dandelion extract

[0046] 1.3.1 Single-factor experiment

[0047] (1) Effect of ethanol concentration on the extraction rate of dandelion extract

[0048] Referring to the extraction process in Example 1, the ultrasonic time was selected as 30 min, the ultrasonic temperature as 80 °C, the liquid-to-solid ratio as 10:1, and the ultrasonic power as 490 W. The changes in EGP extraction rate were investigated when the ethanol concentration was 40%, 55%, 70%, 85%, and 100% (ethanol solutions of different concentrations were prepared by mixing anhydrous ethanol and pure water in proportion).

[0049] (2) Effect of ultrasonic temperature on the extraction rate of dandelion extract

[0050] Referring to the extraction process in Example 1, the ethanol concentration was selected as 70%, the ultrasonic power as 490W, the ultrasonic time as 30min, and the liquid-to-solid ratio as 10:1. The changes in EGP extraction rate were investigated at ultrasonic temperatures of 20, 35, 50, 65, and 80℃.

[0051] (3) Effect of liquid-to-solid ratio on the extraction rate of dandelion extract

[0052] Referring to the extraction process in Example 1, the ethanol concentration was selected as 70%, the ultrasonic power as 490W, the ultrasonic time as 30min, and the ultrasonic temperature as 80℃. The changes in EGP extraction rate were investigated when the liquid-to-solid ratio was 10:1, 20:1, 30:1, 40:1, and 50:1.

[0053] (4) Effect of ultrasonic power on the extraction rate of dandelion extract

[0054] Referring to the extraction process in Example 1, the ethanol concentration was selected as 70%, the ultrasonic time was 30 min, the ultrasonic temperature was 80℃, and the liquid-to-solid ratio was 10:1. The changes in EGP extraction rate were investigated when the ultrasonic power was 280, 350, 420, 490, and 560 W.

[0055] (5) Effect of ultrasonic time on the extraction rate of dandelion extract

[0056] Referring to the extraction process in Example 1, the ethanol concentration was selected as 70%, the ultrasonic power as 490W, the ultrasonic temperature as 80℃, and the liquid-to-solid ratio as 10:1. The changes in EGP extraction rate were investigated when the ultrasonic time was 30, 60, 90, 120, and 150 min.

[0057] Results: The extraction efficiency of the ethanol extract of *Datura stramonium* was determined by the yield of chlorogenic acid. The effects of specific factors are detailed below. Figure 2 The effect of ethanol concentration on EGP extraction rate is shown in [reference needed]. Figure 2 A. The concentration varies from 40% to 100%. The yield of chlorogenic acid increases continuously with the increase of ethanol concentration, reaching a maximum of 1.90±0.01% at a concentration of 70%, and then begins to decrease.

[0058] The effect of ultrasonic power is shown in Figure 2 B. The yield of chlorogenic acid generally increased and then decreased with the change of ultrasonic power, with the highest yield of 1.70±0.01% at 420W.

[0059] Figure 2 As shown in C, the yield of chlorogenic acid first increases and then decreases with the increase of the liquid-to-solid ratio, reaching 1.90±0.01% at 20:1.

[0060] Figure 2 As shown in Figure D, within the ultrasonic temperature range of 20–35℃, the yield of chlorogenic acid gradually increases with increasing power. However, within the ultrasonic temperature range of 35–80℃, the yield of chlorogenic acid begins to decrease with increasing power, and at 35℃, the yield of chlorogenic acid is 1.85 ± 0.02%.

[0061] Figure 2 E represents the effect of ultrasound time on chlorogenic acid yield, showing a trend of first increasing and then decreasing. The highest chlorogenic acid yield (1.78 ± 0.01%) was achieved when the ultrasound time was 60 min.

[0062] 1.3.2 Plackett-Burman Test

[0063] Based on 1.3.1, a Plackett-Burman experiment with N=12 was designed using Minitab 17 software to observe the significance of the above five factors, as shown in Table 3.

[0064] Table 3. Factor Levels in the Plackett-Burman Experiment

[0065]

[0066] Further analysis of the significance of each factor was conducted. Table 4 shows the Plackett-Burman experimental results, and Table 5 shows the results of the analysis of variance. Table 5 shows that the model's P < 0.01, therefore, the model is highly significant. The effects of ethanol concentration (A), liquid-to-solid ratio (C), and ultrasonic power (D) on the yield of chlorogenic acid were all significant (P < 0.05), but the effects of ultrasonic temperature (B) and ultrasonic time (E) were not significant (P > 0.05). Therefore, these three factors were selected for the subsequent response surface methodology optimization experiment, with the ultrasonic temperature fixed at 35℃ and the ultrasonic time fixed at 60 min.

[0067] Table 4. Plackett-Burman Experimental Design and Results

[0068]

[0069]

[0070] Table 5. Significance analysis of each factor in the Plackett-Burman test.

[0071]

[0072] R² = 93.10%, R² Adj = 87.34%, * indicates a significant difference (P<0.05), ** indicates an extremely significant difference (P<0.01)

[0073] 1.3.3 Response Surface Optimization

[0074] Based on 1.3.2, the Box-Behnken response surface methodology was used for optimization to determine the optimal values ​​of significant factors such as ethanol concentration, ultrasonic power, and liquid-to-solid ratio. The variance was analyzed using Design-Expert 8.0.6 software. The specific experimental design is shown in Table 6.

[0075] Table 6. Factor Levels in Box-Behnken Response Surface Experiment

[0076]

[0077]

[0078] Results: (1) Response test results and analysis of variance

[0079] Table 7 shows the response surface methodology results of EGP, yielding the regression model: Y = 3.13 + 0.45A + 0.27B + 0.36C - 0.14AB - 0.15AC - 0.098BC - 0.033A 2 -0.14B 2 -0.13C 2 It can be seen that in this model, A (ethanol concentration), B (ultrasonic power), C (liquid-to-solid ratio), AB, AC, and B... 2 and C 2 The items had a highly significant effect on the yield of chlorogenic acid (P<0.01), while items B and C had a significant effect (P<0.05).

[0080] Furthermore, the model's p-value < 0.01 and the p-value for the lack-fit term > 0.05, indicating that the model is reliable. The goodness of fit Rfit 2 =99.22%, indicating that the fitted value is significantly correlated with the actual value, and the model accuracy meets the requirements. The correction coefficient is 98.23%, indicating that 98.23% of the change in the response value can be explained by the model.

[0081] In conclusion, the above-mentioned model for predicting EGP extraction process is feasible.

[0082] Table 7 Box-Bachnken response surface methodology and results

[0083]

[0084]

[0085] Table 8. Results of ANOVA for the Regression Model

[0086]

[0087] R 2 =99.22%, R 2 Adj = 98.23%, * indicates a significant difference (P<0.05), ** indicates an extremely significant difference (P<0.01)

[0088] (2) Response interaction analysis: The influence of the interaction between various factors on the response value is shown in [see...] Figure 3 As can be seen, the 3D response surface plots are all parabolic, with a maximum point. Combined with... Figure 3As shown in Table 8, AC has the steepest response surface curve and the greatest impact on chlorogenic acid yield. The order of influence of the interaction on chlorogenic acid yield in EGP is: AC > AB > BC.

[0089] (3) Optimal Condition Verification

[0090] Using Design-Expert 8.0.6 software, model analysis revealed the optimal conditions for ultrasonic-assisted extraction of dandelion powder as follows: ethanol concentration 85%, ultrasonic power 434.15W, liquid-to-solid ratio 27.52:1, ultrasonic temperature 35℃, and ultrasonic time 60 min. Under these conditions, the theoretical yield of chlorogenic acid in dandelion powder can reach 3.64%. For ease of operation, the optimal extraction conditions were further defined as: ethanol concentration 85%, ultrasonic power 430W, liquid-to-solid ratio 25:1, ultrasonic temperature 35℃, and ultrasonic time 60 min.

[0091] Three verification experiments were conducted under the modified optimal conditions. The yield of chlorogenic acid in dandelion powder reached 3.51±0.09%, which is very close to the predicted value, indicating that the extraction process optimized by response surface methodology is feasible. Figure 4 The appearance of the lyophilized extract of fentanyl obtained from the optimal extraction process is that it is in powder form.

[0092] Example 3

[0093] 1. Infrared spectroscopy analysis

[0094] Weigh 1.0 mg of lyophilized EGP powder and 300.0 mg of potassium bromide (KBr), mix them thoroughly in an agate mortar and grind them into powder, then prepare transparent tablets under vacuum at 400–4000 cm⁻¹. -1 The pellet was scanned using a Fourier transform infrared spectrometer within a certain range, with a resolution of 1 cm⁻¹. -1 .

[0095] Results: The infrared spectral scan results of the ethanol extract of dandelion are as follows: Figure 5 A comparison revealed that the absorption peaks and peak shapes of the two were very similar. Specifically, EGP had a peak at 3365 cm⁻¹. -1 The broad peak at 2926 cm⁻¹ is a -OH stretching vibration peak, with a wide peak shape and extremely strong absorption, indicating the presence of phenolic hydroxyl groups or hydroxyl groups on sugars; EGP at 2926 cm⁻¹ -1 The peak at 1608 cm⁻¹ is the CH stretching vibration peak; the peak at 1608 cm⁻¹ is the EGP peak. -1 1385cm -1 The relatively strong absorption peak at 1268 cm⁻¹ corresponds to the stretching vibration of phenolic double bonds; EGP is at 1268 cm⁻¹. -1 and 1049cm -1 The absorption peak at 606 cm⁻¹ corresponds to the stretching vibration of COC; the EGP peak at 606 cm⁻¹ corresponds to the stretching vibration of COC. -1The absorption peak at that location is caused by the out-of-plane bending vibration of the CH group on the benzene ring. Based on the above peak characteristics, it can be inferred that the dandelion extract may contain flavonoids, phenols, or phenylpropanoids; further analysis is needed to determine the specific composition.

[0096] 2. UHPLC-QE-MS analysis of non-target metabolomics of traditional Chinese medicine

[0097] (1) Sample preparation

[0098] Centrifuge the dandelion extract or alcohol extract at 4℃ and 12000rpm for 15 min, and collect 300μL of the supernatant into an EP tube. Add 1000μL of extraction buffer (methanol:water = 4:1, internal standard concentration of 10μg / mL). Sonicate the mixture in an ice-water bath for 5 min, then let it stand at -40℃ for 1 h. Centrifuge the sample at 4℃ and 12000rpm for 15 min, collect the supernatant, filter it through a 0.22μm filter membrane, and then place it in a sample vial for instrumental analysis. Mix 100μL of each sample to form a QC sample.

[0099] (2) Chromatographic conditions

[0100] A UPLC BEHC18 column (2.1*100mm, 1.7μm) was used, with a flow rate of 0.4mL / min and an injection volume of 5μL. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B). The elution gradient was: 0–3.5 min, 95–85% A; 3.5–6 min, 85–70% A; 6–6.5 min, 70–70% A; 6.5–12 min, 70–30% A; 12–12.5 min, 30–30% A.

[0101] (3) Mass spectrometry conditions

[0102] An Orbitrap Exploris 120 mass spectrometer and Xcalbur software were used for MS and MS / MS data acquisition in IDA mode. Specific parameters were as follows: sheath gas flow rate 30 arb, auxiliary gas flow rate 10 arb, ion transfer tube and evaporator temperatures 350 °C, full millisecond resolution 60,000, MS / MS resolution 15,000, collision energy 16 / 38 / 42, and spray voltage 5.5 kV (positive ion mode) or 4 kV (negative ion mode).

[0103] (4) Data processing

[0104] The raw mass spectrometry data was imported using Progenesis QI software. At the same time, a metabolic library of the corresponding Chinese herbal medicines in the compound was created. The substances containing MSMS data were identified by using a self-built secondary mass spectrometry database and the corresponding fragmentation pattern matching method.

[0105] 2.1 Identification of non-target metabolomics components of Dandelion extract

[0106] To further analyze the specific material composition of the ethanol extract of dandelion, it was identified by UHPLC-QE-MS non-target metabolomics, and the top 40 components with the highest scores were listed in Table 9.

[0107] Table 9. Component Analysis of Dandelion Extract

[0108]

[0109]

[0110]

[0111]

[0112] Note: NEG indicates negative ion mode, and POS indicates positive ion mode.

[0113] It is known that the ethanol extract of dandelion contains various compounds, such as phenols (Ⅰ), terpenes (Ⅱ), alkaloids (Ⅲ), organic acids and their derivatives (Ⅳ), flavonoids (Ⅴ), phenylpropanoids (Ⅵ), benzoic acid and its derivatives (Ⅶ), amino acid derivatives (Ⅷ), organic oxygen compounds (Ⅸ), sesquiterpenes (Ⅹ), chalcones (ⅩⅠ), quinones (ⅩⅡ), other compounds (ⅩⅢ), and isopentenyl esters (ⅩⅣ). The ethanol extract contains higher levels of 14 components, including rhodioloside, fraxin, quercetin, apigenin, and others. However, analysis of the filter residue of the dandelion ethanol extract revealed that it still contained many unextracted active substances. Therefore, this invention also explored the extraction process of active substances from the filter residue of the ethanol extract.

[0114] Example 4

[0115] A method for preparing dandelion extract, comprising the following steps:

[0116] (1) The dandelion powder was ground into 80 mesh using a grinder (60g dandelion, 60g honeysuckle, 60g forsythia, 30g loofah, 25g tetrapanax papyriferus, 25g hibiscus leaves, 30g Fritillaria thunbergii). The powder was soaked in 85% ethanol for 30 min at a liquid-to-solid ratio of 25:1 (v / w). Then, it was extracted using an ultrasonic extractor at 35℃ and 430W for 60 min. After extraction, the mixture was cooled to room temperature and centrifuged at 3000 rpm for 10 min to obtain the supernatant and filter residue.

[0117] (2) The filter residue was mixed with water with pH 8.2 at a liquid-to-solid ratio of 1g:12mL and then subjected to subcritical extraction to obtain the filtrate; the temperature of the subcritical extraction was 120℃ and the pressure was 2MPa; the time of the subcritical extraction was 30min.

[0118] (3) The alcohol extract and the filtrate were concentrated and dried separately and then combined to obtain dandelion extract.

[0119] Example 5

[0120] A method for preparing dandelion extract, comprising the following steps:

[0121] (1) The dandelion powder was ground into 80 mesh using a grinder (60g dandelion, 60g honeysuckle, 60g forsythia, 30g loofah, 25g tetrapanax papyriferus, 25g hibiscus leaves, 30g Fritillaria thunbergii). The powder was soaked in 85% ethanol for 30 min at a liquid-to-solid ratio of 25:1 (v / w). Then, it was extracted using an ultrasonic extractor at 35℃ and 430W for 60 min. After extraction, the mixture was cooled to room temperature and centrifuged at 3000 rpm for 10 min to obtain the supernatant and filter residue.

[0122] (2) The filter residue was mixed with water with pH 10.2 at a liquid-to-solid ratio of 1g:16mL (v / w) and then subjected to subcritical extraction to obtain the filtrate; the temperature of the subcritical extraction was 210℃ and the pressure was 4MPa; the time of the subcritical extraction was 70min.

[0123] (3) The alcohol extract and the filtrate were concentrated and dried separately and then combined to obtain dandelion extract.

[0124] Example 6

[0125] A method for preparing dandelion extract, comprising the following steps:

[0126] (1) The dandelion powder was ground into 80 mesh using a grinder (60g dandelion, 60g honeysuckle, 60g forsythia, 30g loofah, 25g tetrapanax papyriferus, 25g hibiscus leaves, 30g Fritillaria thunbergii). The powder was soaked in 85% ethanol for 30 min at a liquid-to-solid ratio of 25:1 (v / w). Then, it was extracted using an ultrasonic extractor at 35℃ and 430W for 60 min. After extraction, the mixture was cooled to room temperature and centrifuged at 2500 rpm for 10 min to obtain the supernatant and filter residue.

[0127] (2) The filter residue was mixed with water at pH 9.2 at a liquid-to-solid ratio of 1g:14mL (v / w) and then subjected to subcritical extraction to obtain the filtrate; the temperature of the subcritical extraction was 180℃ and the pressure was 3MPa; the time of the subcritical extraction was 50min.

[0128] (3) The alcohol extract and the filtrate were concentrated and dried separately and then combined to obtain dandelion extract.

[0129] Example 7

[0130] The only difference from Example 3 is that the pH of the water is set to 7.0.

[0131] Example 8

[0132] The only difference from Example 3 is that the subcritical extraction pressure is 6 MPa.

[0133] Example 9

[0134] The only difference from Example 3 is that the subcritical extraction temperature is 230°C.

[0135] Following the method in step 1 of Example 2, the content of chlorogenic acid obtained by the methods in Examples 6 to 9 was determined, and the results are shown in Table 10.

[0136] Table 10 Chlorogenic Acid Content

[0137] chlorogenic acid content Example 4 4.97％ Example 5 5.01％ Example 6 5.12％ Example 7 4.82％ Example 8 4.23％ Example 9 4.41％

[0138] Table 10 shows that different extraction methods significantly affect the chlorogenic acid content in the extract. The method in Example 6 yielded the highest chlorogenic acid content, reaching 5.12%, followed by the methods in Examples 5 and 4. This content far exceeds that of the chlorogenic acid in the ethanol extract prepared in Example 1, indicating that the filter residue of the ethanol extract still contains many active substances that have not been extracted. Based on the data, the dandelion extract prepared by the method in Example 6 is suitable for further research.

[0139] The data from the control group show that the subcritical extraction method used in this invention can further extract the active substances in the filter residue, thereby improving the extraction efficiency.

[0140] Example 10

[0141] 1. Preparation of test bacterial suspension

[0142] Test strains: Bovine Escherichia coli (E. coli) CVCC 1450, Streptococcus agalactis (S. agalactiae) CVCC 3940, and Staphylococcus aureus (S. aureus) ATCC 29740 were purchased from the China Veterinary Microbiological Culture Collection Center.

[0143] E. coli CVCC 1450, S. agalactiae CVCC 3940, and S. aureus ATCC 29740 were cultured overnight to the logarithmic growth phase, and the test bacterial cultures were diluted 10-fold with sterile broth. -5 10 -6 10 -7 10 -8 Different concentrations were obtained, and then 50 μL of the diluted bacterial solution from the test tube was dropped onto the surface of the solid culture medium. After incubation at 37°C for 24 hours, colony counting was performed, and finally 5 × 10⁶ colonies were prepared. 5 CFU / mL and 5×10 8 The bacterial culture at CFU / mL was used for subsequent experiments.

[0144] Preparation of Dandelion Extract Stock Solution: Accurately weigh 5.76 g of EGP lyophilized powder and dissolve it in 10 mL of 30% ethanol solution to prepare a 576 mg / mL ethanol extract stock solution. Sterilize the dandelion extract stock solution by filtration through a 0.22 μm filter membrane, and then perform serial two-fold dilutions with sterile broth to obtain test sample solutions with different concentrations of 288, 144, 72, 36, 18, 9, 4.5, 2.25, 1.13, and 0.56 mg / mL.

[0145] Preparation of Dandelion Extract Solution: Accurately weigh 5.76 g of the Dandelion extract prepared in Example 6 and dissolve it in 10 mL of 30% ethanol solution to obtain a Dandelion extract stock solution with a concentration of 576 mg / mL. The Dandelion ethanol extract stock solution was sterilized by filtration through a 0.22 μm filter membrane, and then serially diluted twofold with sterile broth culture medium to obtain test sample solutions with different concentrations of 288, 144, 72, 36, 18, 9, 4.5, 2.25, 1.13, and 0.56 mg / mL.

[0146] 3. Determination of the minimum inhibitory concentration and minimum bactericidal concentration of dandelion extract.

[0147] Add 100 μL of the prepared test solutions of different concentrations from step 2 to the first 10 wells of the 96-well plate, followed by 100 μL of the test bacterial suspension (5 × 10⁻⁶). 5 The last two wells were positive (0.20 mL bacterial suspension) and negative (0.20 mL broth) control wells. The 96-well plates were incubated at 37°C for 24 hours. The last clear well showing no obvious bacterial growth was the minimum inhibitory concentration (MIC), and the last well showing no bacterial growth when dropped onto solid medium was the minimum bactericidal concentration (MBC). Each experiment was repeated three times.

[0148] 4. Determination of the diameter of the inhibition zone of dandelion extract

[0149] Using the Kirby-Bauer disc diffusion method, 0.10 mL of 5 × 10⁻⁶ solution was used. 8 The bacterial suspension of CFU / mL was evenly spread on MH(A) medium, and then antibiotic discs (6.00 mm) containing penicillin, cephalexin, vancomycin, and gentamicin, as well as discs containing high concentration (144.00 mg / mL) and low concentration (36.00 mg / mL) dandelion extract (or EGP) were attached. The mixture was incubated overnight at 37°C for 24 h, and the diameter of the inhibition zone was measured.

[0150] 5. Test Results

[0151] The results in Tables 11 and 12 show that the dandelion extract prepared in Example 6 exhibited the best antibacterial effect against E. coli CVCC 1450, E. coli CVCC 1450, and S. agalactiae CVCC 3940. This antibacterial effect was significantly better than that of the dandelion alcohol extract.

[0152] Table 11. In vitro antibacterial results of EGP (mg / mL)

[0153]

[0154] Table 12. In vitro antibacterial results (mg / mL) of Example 6

[0155]

[0156] 5.2 Diameter of the inhibition zone of dandelion extract

[0157] The results showed that the extract prepared in Example 6 had a relatively good antibacterial effect against Gram-negative E. coli CVCC 1450, and the best antibacterial effect against Gram-positive S. aureus ATCC 29740 and S. agalactiae CVCC 3940. This antibacterial effect was significantly better than that of the dandelion extract, indicating that the subcritical extraction method of the present invention further extracted the active ingredients from the dandelion, further improving the efficacy of the drug.

[0158] Table 13. Diameter of the inhibition zone of EGP (X±SD, mm)

[0159]

[0160]

[0161] "—" indicates no inhibition zone, and " / " indicates that the test was not conducted.

[0162] Table 14. Diameter of the inhibition zone (X±SD, mm) in Example 6

[0163]

[0164] "—" indicates no inhibition zone, and " / " indicates that the test was not conducted.

[0165] Example 11

[0166] 1. DPPH free radical scavenging test

[0167] Prepare a 0.20–1.20 mg / mL Vc solution as a positive control, and prepare a 0.50–3.00 mg / mL extract (or EGP) prepared in Example 6 as a test sample. Accurately transfer 1.00 mL of each of the test sample and DPPH-anhydrous ethanol solution of different concentrations into test tubes, mix well, react in the dark for 30 min, and detect the absorbance of the mixed solution at a wavelength of 517 nm. Calculate the clearance rate according to formula (2).

[0168] DPPH free radical scavenging rate = [1-(A Ⅰ -A Ⅱ ) / A Ⅲ ]×100% Formula (2)

[0169] In the formula: A represents the absorbance of different solutions (Ⅰ is the sample; Ⅱ is the anhydrous ethanol solution; Ⅲ is the blank control).

[0170] 2. Hydroxyl radical scavenging test: The hydroxyl radical scavenging rate was determined by the salicylic acid method. 2.00 mL of each of the different test samples, ferrous sulfate solution (9.00 mmol / L), salicylic acid-anhydrous ethanol solution (9.00 mmol / L), and hydrogen peroxide solution (8.80 mmol / L) were accurately transferred into test tubes and mixed. The mixture was then reacted at 37 °C for 30 min. The absorbance of the mixed solution was measured at a wavelength of 510 nm, and the scavenging rate was calculated according to formula (3).

[0171] Hydroxyl radical scavenging rate = [1-(A Ⅰ -A Ⅱ ) / A Ⅲ ]×100% formula (3)

[0172] In the formula: A represents the absorbance of different solutions (Ⅰ is the sample; Ⅱ is the hydrogen peroxide solution; Ⅲ is the blank control).

[0173] Table 15 DPPH Free Radical Scavenging Test

[0174]

[0175] Table 16 Hydroxyl radical scavenging test

[0176]

[0177]

[0178] Results: The dandelion extract prepared in Example 6 showed higher DPPH scavenging ability than the ethanol extract, but lower than the positive control (Vc). All three showed concentration-dependent DPPH free radical scavenging rates within the range of 0.20–1.00 mg / mL.

[0179] It can be seen that the DPPH scavenging ability, from high to low, is as follows: vitamin C, dandelion extract, and EGP. Although the scavenging ability of dandelion extract against hydroxyl radicals is not as good as that of the positive control (vitamin C), it still has a good scavenging efficiency when the concentration increases.

[0180] The above description is only a preferred embodiment of the present invention.

Claims

1. A method for preparing a ginseng extract, characterized by, The method comprises the following steps: (1) crushing Gongying Powder and mixing with ethanol with a volume fraction of 80-90% to soak, extracting by ultrasonic, and obtaining alcohol extract and residue by centrifugation; (2) mixing the residue with water and obtaining filtrate by subcritical extraction; (3) concentrating and drying the alcohol extract and the filtrate respectively and then combining to obtain Gongying Powder extract; In step (1), the temperature of ultrasonic is set to 30-90℃, the power of ultrasonic is set to 350-450W, and the mass-volume ratio of Gongying Powder to ethanol is 1g:20-40mL; In step (2), the mass-volume ratio of residue to water is 1g:12-16mL, the temperature of subcritical extraction is 120-210℃, and the pressure is 2-4MPa; Gongying Powder is composed of the following components by mass: Herba Taraxaci 60g, Flos Lonicerae 60g, Fructus Forsythiae 60g, Retinervus Luffae Fructus 30g, Herba Veratri 25g, Folium Hibisci 25g, and Bulbus Liliacearum 30g.

2. The preparation method of the Gongseng extract according to claim 1, characterized in that, In step (1), the soaking time is 25-35min.

3. The method of claim 1, wherein the preparation of the extract of Radix Angelicae Pubescentis and Radix Angelicae Sinensis is characterized by, In step (1), the ultrasonic time is set to 50-70min.

4. The preparation method of the Gongseng extract according to claim 1, characterized in that, In step (1), the centrifugation speed is 2000-3000rpm, and the centrifugation time is 8-12min.

5. The method of claim 1, wherein the preparation of the extract of Radix Angelicae Pubescentis and Radix Angelicae Sinensis is characterized by, In step (2), the pH of water is 8.2-10.2, and the subcritical extraction time is 30-70min.

6. Gongying Powder extract prepared by the preparation method of Gongying Powder extract according to any one of claims 1-5.

7. Application of Gongying Powder extract according to claim 6 in preparing an inhibitor for bovine mastitis.

8. Use according to claim 7, characterized in that, The pathogenic bacteria of bovine mastitis are one or more of Staphylococcus aureus, Escherichia coli, or Streptococcus agalactiae.

9. Application of Gongying Powder extract according to claim 6 in preparing an antioxidant.

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