Crocus sativus lateral bud flavone extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of natural product extraction and separation, and particularly relates to a saffron lateral bud flavone extract as well as a preparation method and application thereof. The method specifically comprises the following steps: S.1, crushing dried lateral buds of saffron by using a crusher, sieving, weighing powder, extracting by using an ultrasonic circulating extraction method, and filtering an extracting solution to obtain supernate; and S.2, carrying out vacuum concentration on the supernatant to obtain an extract, and carrying out freeze drying treatment to obtain the final extract. The ultrasonic circulating extraction technology adopted by the invention has the advantages of simplicity and convenience in operation, high extraction efficiency, short time consumption and the like, and can realize efficient extraction of the flavone component in the lateral buds of the saffron. The extract shows good inhibition activity on aspergillus flavus and aspergillus parasiticus, which indicates that the extract has potential application value in the antibacterial field. The process is suitable for laboratory small-scale preparation and industrial production, and has good popularization and application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product extraction and separation, and particularly relates to a crocus sativus L. lateral bud flavone extract as well as a preparation method and application thereof. BACKGROUND

[0002] Crocus sativus L. is a kind of perennial bulbous plant in Iridaceae, and is also called saffron. Crocus sativusL.)is a kind of medicinal and edible precious traditional Chinese medicine, usually with dry stigma as medicine, which has the effects of promoting blood circulation to remove blood stasis, cooling blood to detoxify, relieving depression and soothing the nerves. It is often used in clinical treatment of postpartum blood stasis, palpitation and mania. In many countries, saffron is also widely used as a traditional medicine and natural spice. Because of its significant economic value and broad market prospects, many countries around the world continue to carry out research and development of its cultivation technology and active ingredients. In order to improve the yield and quality of saffron, the lateral buds of the plant need to be removed during cultivation, and the yield is closely related to the size of the mother corm. Generally, it is recommended to leave one bud for corms less than 16 g, two buds for corms between 16 and 25 g, and three buds for corms greater than 25 g. However, the quality of these discarded lateral buds is much higher than the average stigma yield, but they are often discarded in saffron cultivation, causing great waste of resources. Studies have shown that saffron lateral buds contain flavonoids, anthraquinones, polysaccharides and other active ingredients, which have high development and utilization value. Wang et al. used silica gel column chromatography to preliminarily separate the ethyl acetate phase of saffron lateral bud extract, and combined with HPLC-MS / MS to analyze its main chemical components. The results showed that polyphenolic compounds were the main active ingredients (Industrial Crops and Products, 2021, 173: 114081). Gao et al. isolated two new phenolic glucosides, a new γ-lactone glucoside and adenosine from the n-butanol extract of saffron lateral buds; based on chemical and spectral data, the structures were identified as 2,4-dihydroxy-6-methoxy acetylacetone-2β-D-glucopyranoside (1), 2,3,4-trihydroxy-6-methyl acetyl ketone-3-β-D-glucopyranoside (2) and 3-(S)-3-β-D-glucopyranosyl acyloxy butyrolactone (3) (Planta Medica, 1999, 65(5): 425-427). Gao et al. isolated four anthraquinones from the chloroform extract of saffron lateral buds, and determined their chemical structures by spectral and chemical methods as emodin and 2-hydroxy emodin, and two new anthraquinones: 1-methyl-3-methoxy-8-hydroxy anthraquinone-2-carboxylic acid and 1-methyl-3-methoxy-6,8-dihydroxy anthraquinone-2-carboxylic acid (Acta Botanica Sinica, 1999, 41(5): 531-533). Rao et al. used water extraction and alcohol precipitation method to extract crude polysaccharides from saffron lateral buds, and used ion chromatography-pulse amperometric method to determine the monosaccharide composition of polysaccharides; under the optimized conditions, seven kinds of monosaccharides were detected in the lateral bud polysaccharides, including fucose, rhamnose, arabinose, mannose, glucose, galactose and fructose (Journal of Zhejiang University (Science), 2015, 42(03): 330-333).In addition, L-citrulline is rich in safflower lateral buds, and the content is about 26.69 mg / g after optimization of the extraction process (Journal of Sichuan University (Natural Science Edition), 2018, 55(6): 1297-1300).

[0003] The ultrasonic circulation extraction technology is a high-efficiency extraction method combining ultrasonic wave action and continuous circulation system. The core is to accelerate the dissolution of target components in materials by ultrasonic cavitation effect and circulation process, improve the extraction efficiency, and be beneficial to the stable retention of heat-sensitive components. Compared with the traditional extraction process, the technology performs better in high extraction efficiency and component protection. At present, there is no related report about the ultrasonic circulation extraction technology for extracting and preparing flavonoids in safflower lateral buds. SUMMARY

[0004] In view of the problems in the prior art, the purpose of the present application is to design and provide a safflower lateral bud flavone extract and a preparation method and application thereof.

[0005] In a first aspect, a preparation method of a safflower lateral bud flavone extract is provided, which specifically comprises the following steps: S.1, dry safflower lateral buds are crushed by a crusher and sieved, the powder is weighed, and ultrasonic circulation extraction is performed, and the supernatant is obtained after the extraction liquid is filtered; S.2, the supernatant is reduced pressure concentrated into extract, and the final extract is obtained by freeze-drying treatment.

[0006] Further, the ultrasonic circulation extraction conditions in step S.1 are as follows: ultrasonic time is 30-50 min, ultrasonic power is 400-800 W, and stirring frequency is 800-1200 rpm.

[0007] Further, the temperature of ultrasonic circulation extraction in step S.1 is 30-50℃.

[0008] Further, ethanol is used for ultrasonic circulation extraction in step S.1, and the concentration of the ethanol is 40-80%.

[0009] Further, the solid-liquid ratio of ultrasonic circulation extraction in step S.1 is sample: ethanol = 20-40 mL / g.

[0010] In a second aspect, the present application provides a safflower lateral bud flavone extract.

[0011] In a third aspect, the present application provides the application of the safflower lateral bud flavone extract in preparing antibacterial products.

[0012] Further, the antibacterial is inhibiting Aspergillus flavus and Aspergillus parasiticus.

[0013] The present application has the following beneficial effects: (1) The ultrasonic circulation extraction technology adopted in the present application has the advantages of simple operation, high extraction efficiency and short time consumption, and can realize efficient extraction of flavone components in crocus lateral buds.

[0014] (2) The extract obtained in the present application shows good inhibitory activity on Aspergillus flavus and Aspergillus parasiticus, indicating that it has potential application value in the field of antibiosis.

[0015] (3) The process of the present application is suitable for small-scale preparation in the laboratory and industrial production, and has good popularization and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a response surface analysis diagram; A, C, E are response surface diagrams of the interaction between the three factors of RSM, and B, D, F are contour diagrams; Figure 2 is the effect of crocus lateral bud extract treatment on the change of Aspergillus flavus colony diameter; Figure 3 Figure 3 is the antibacterial effect of crocus lateral bud extract on Aspergillus flavus; Figure 4 Figure 4 is the effect of crocus lateral bud extract treatment on the change of Aspergillus parasiticus colony diameter; Figure 5 Figure 5 is the antibacterial effect of crocus lateral bud extract on Aspergillus parasiticus. DETAILED DESCRIPTION

[0017] The present application will be further described below in combination with specific embodiments.

[0018] All the following tests use spectrophotometry to determine the flavone content.

[0019] The preparation method of the standard curve is as follows: accurately weigh the rutin reference substance, dissolve and prepare a reference substance solution with a concentration of 453.8 µg / mL. Accurately take 0.1, 0.2, 0.25, 0.3, and 0.4 mL of the reference substance solution into a 1 mL volumetric flask, dilute to the mark with an appropriate amount of anhydrous ethanol, and shake well. Then add 0.1 mL of 5% sodium nitrite solution, mix thoroughly, and react for 6 min; then add 0.1 mL of 10% aluminum nitrate solution, mix well, and continue to react for 6 min; finally, add 0.8 mL of 4% sodium hydroxide solution, mix thoroughly, and react for 15 min. Measure the absorbance of each solution at 510 nm using an enzyme marker. Take anhydrous ethanol as a blank reference, take the concentration of rutin solution (µg / mL) as the abscissa x, and the absorbance (A) as the ordinate y, to draw a standard curve, and obtain the linear regression equation: y = 0.004x + 0.0936 (R 2 = 0.9995).

[0020] Sample test: The croci lateral bud extract prepared by the above ultrasonic circulation extraction was dissolved and diluted with an appropriate amount of ethanol, and then the absorbance was measured according to the same determination steps as the standard curve. The flavone concentration was calculated according to the standard curve, and the flavone content of the sample extract was further calculated.

[0021] Example 1: Plackett-Burman experimental design and result analysis S.1, The dried croci lateral bud was crushed by a pulverizer and passed through a 60-mesh sieve. The powder was weighed and extracted by ultrasonic circulation extraction. The extract was filtered to obtain supernatant; S.2, The supernatant was reduced pressure concentrated to extract, and then freeze-dried to obtain the final extract.

[0022] In the ultrasonic circulation extraction process, six factors, i.e. ultrasonic time (A), ultrasonic temperature (B), solid-liquid ratio (C), ultrasonic power (D), ethanol concentration (E) and stirring frequency (F) were selected as single factor variables. The Plackett-Burman experimental design was used to carry out the key influencing factor screening experiment. The total flavone extraction amount of croci lateral bud was used as the response index. Each factor was set at two levels, represented by 1 and -1, respectively. A total of 12 experiments were designed. The factor levels and schemes are shown in Tables 1 and 2.

[0023] Table 1 Factor level table of Plackett-Burman design

[0024] Table 2 Plackett-Burman experimental design and results

[0025] The experimental results were analyzed by stepwise regression analysis using Design expert 13.0 software. The regression equation for the total flavone extraction amount as the response value was: total flavone extraction amount = 2.69-0.1024A+0.2554B+0.1499C+0.0110D+0.2483E-0.1080F.

[0026] According to the variance analysis results in Table 3, the P value of the overall model is 0.0088, which is less than 0.05, indicating that the regression model has statistical significance. Among them, the ultrasonic temperature, solid-liquid ratio and ethanol concentration have a significant effect on the total flavone extraction amount, while the ultrasonic time, power and stirring frequency have no significant effect on the total flavone extraction amount. Therefore, the ultrasonic temperature, ethanol concentration and solid-liquid ratio are the main influencing factors of the ultrasonic circulation extraction process.

[0027] Table 3 Variance analysis of Plackett-Burman experimental design

[0028] Example 2: Response surface experiment design and result analysis According to the screening results of the Plackett-Burman experiment, the ultrasonic temperature, the solid-liquid ratio, and the ethanol concentration were selected as the independent variables, the ultrasonic time was fixed at 30 min, the ultrasonic power was fixed at 800 W, the stirring frequency was fixed at 800 rpm, the total flavonoid extraction yield was taken as the response value, and the response surface analysis method with three factors and three levels was used for the research according to the Box-Benhnken central composite experiment design principle. The factor level coding is shown in Table 4, the experiment design scheme and the results are shown in Table 5.

[0029] Table 4 Factor level table of Box-Behnken design

[0030] Table 5 Box-Behnken design scheme and response value

[0031] (1) Model construction and variance analysis The experiment data in Table 5 were regressed and fitted by using the Design expert 13.0 software, and a quadratic multinomial regression equation of the three independent variables was obtained.

[0032] The variance analysis results of the regression model are shown in Table 6. The results show that A, B, C, and their mutual terms AB, AC, BC, A 2 , B 2 , C 2 all have a very significant influence on the total flavonoid extraction yield of Crocus sativus L. (P < 0.01). The total determination coefficient R 2 = 0.9939, the corrected determination coefficient R Adj 2 = 0.9816, and the predicted determination coefficient R Pred 2 = 0.9179, and the variation coefficient CV is 1.45%, indicating that the multivariate regression relationship between the dependent variable and each independent variable is highly significant, the model fitting condition is good, and the experiment error is small. Therefore, the regression equation can accurately describe the relationship between each factor and the response value, and is suitable for the ultrasonic circulation extraction process analysis and optimization of the total flavonoids of Crocus sativus L.

[0033] Table 6 Variance analysis of regression equation

[0034] (2) Response surface analysis of each factor interaction and prediction verification of the optimal process conditions The response surface and contour plot of the interaction between the three factors according to Box-Benhnken experimental design are shown in Figure 1 Figure 1 , and B in Figure 1 , it can be seen that when the ethanol concentration is fixed at 60%, the total flavone extraction amount gradually increases with the increase of ultrasonic temperature when the solid-liquid ratio remains unchanged; when the ultrasonic temperature remains unchanged, the total flavone extraction amount increases first and then decreases with the increase of the solid-liquid ratio; the 3D surface of the interaction between ultrasonic temperature and solid-liquid ratio has obvious curvature and slope, and the corresponding P value is 0.0006, indicating that the interaction between the two has a significant effect on the total flavone extraction amount in crocus side bud. From C in Figure 1 and D in Figure 1 , it can be seen that when the solid-liquid ratio is fixed at 30 mL / g, the total flavone extraction amount decreases slightly first and then increases significantly with the increase of ethanol concentration when the ultrasonic temperature remains unchanged; when the ethanol concentration remains unchanged, the total flavone extraction amount continuously increases with the increase of ultrasonic temperature, and the results in Table 6 also confirm that the interaction between ultrasonic temperature and ethanol concentration is significant. From E in P and F in Figure 1 , it can be seen that when the ultrasonic temperature is set to 40℃, the total flavone extraction amount first increases significantly and then decreases slightly with the increase of the solid-liquid ratio when the ethanol concentration remains unchanged; when the solid-liquid ratio remains unchanged, the total flavone extraction amount decreases slightly first and then increases significantly with the increase of ethanol concentration; the 3D surface has a certain curvature and slope, indicating that the interaction between the solid-liquid ratio and ethanol concentration has a significant effect on the total flavone extraction amount in crocus side bud. Based on the 3D response surface, contour plot and each interaction Figure 1 value analysis, the significant order of the interaction of AB, AC and BC is: AB>BC>AC. P

[0035] Based on the regression model established by Design expert 13.0 software, the optimal extraction process conditions are predicted as follows: ultrasonic temperature 50℃, solid-liquid ratio 33.3 mL / g, and ethanol concentration 80%. Under these conditions, the predicted value of the total flavone extraction amount in crocus side bud is 3.62 mg / g, and the actual measured value is 3.60±0.35 mg / g (n=3), which is close to the predicted value with small deviation, indicating that the regression simulation fitting effect is good, and the extraction process conditions determined by the response surface method have high accuracy and reliability.

[0036] Example 3: UPLC-MS qualitative analysis of crocus side bud extract ​​The target compounds were separated by Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm) using Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatography. The mobile phase A was 0.01% acetic acid, and the mobile phase B was isopropanol:acetonitrile (1:1, v / v) mixed solvent; the column temperature was 30 ℃, and the injection volume was 2 μL. Mass spectrometry analysis was performed using Orbitrap Exploris 120 mass spectrometer under the control of Xcalibur software (version: 4.4) to collect primary and secondary mass spectrometry data. The detailed parameters of the DDA method are as follows: the sheath gas flow rate was 50 Arb, the auxiliary gas flow rate was 15 Arb, the capillary temperature was 320 ℃; the primary mass spectrometry resolution was 60000, and the secondary mass spectrometry resolution was 15000; the collision energy was set to 20%, 30% and 40% using stepwise normalized collision energy (SNCE); the mass spectrometry voltage in positive and negative ion modes was 3.8 kV and -3.4 kV, respectively.

[0037] Based on the above method, the original data of the crocus sativus extract UPLC-MS detection were converted into mzXML format by ProteoWizard software (V3.0.24054), and the metabolite identification was performed using the R language program package developed in cooperation, and the database was self-built and public database. The results showed that among the detection signals with a relative intensity greater than 10 7 , a total of 74 flavonoid components were preliminarily identified (see Table 7), including kaempferol-3-O-acetyl-rutinoside, kaempferol-3-O-rutinoside, rutin, kaempferol-3-O-glucoside, quercetin-3-O-rutinoside, etc.

[0038] Table 7 Identification of crocus sativus flavonoid components by UPLC-MS

[0039] Example 4: Anti-mold activity of crocus sativus extract The above prepared Crocus sativus L. extract freeze-dried powder was dissolved with 5% dimethyl sulfoxide (DMSO) to prepare a mother liquor with a concentration of 1000 mg / mL. The mycelial growth rate method was used to evaluate the inhibitory activity of Crocus sativus L. extract on Aspergillus flavus and Aspergillus parasiticus. Under sterile conditions, different volumes of extract solution were added to PDA medium at a temperature of about 50°C, mixed thoroughly, and prepared into medium containing extract with a final concentration of 0, 25, 50, 100, and 200 mg / mL, with three replicates for each concentration. After the medium solidified, a puncher was used to cut a uniform diameter and evenly grown fungus cake from the edge of the Aspergillus flavus (or Aspergillus parasiticus) colony in the logarithmic growth phase, inoculated in the center of the medium and inverted for culture. The inoculated plate was placed in a constant temperature incubator at 28°C for 7 days. The colony diameter was measured once every 24 hours, and the data was determined and recorded using the cross method, with the average value of three replicates taken as the result. The treatment without adding extract was used as the control group, and the mycelial growth inhibition rate of different concentrations of extract treatment was calculated, with the inhibition rate calculation formula as follows:

[0040] The experimental results showed that Crocus sativus L. extract treatment had a significant inhibitory effect on the growth of Aspergillus flavus (see Figure 2 、 3 ). With the extension of the culture time, the colony diameter of each treatment group showed a growth trend, but with the increase of the extract concentration, the mycelial growth was significantly inhibited, and the colony diameter decreased. After 7 days of culture, the control group had vigorous colony growth. When the concentration of Crocus sativus L. extract reached 60 mg / mL and above, the growth of the colony was completely inhibited within the first 2 days, and the colony diameter remained at 1.2 cm, with an inhibition rate of 100%; but at the 3rd day, the 60 mg / mL treatment group showed slight mycelial expansion, with a colony diameter of 1.35±0.05 cm and an inhibition rate of 93.50±2.36%. The 80 mg / mL treatment group did not observe mycelial growth during the entire 7-day culture period, with a colony diameter of 1.2 cm and a continuous inhibition rate of 100%. Therefore, under the conditions of this experiment, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of Crocus sativus L. extract on Aspergillus flavus were 60 mg / mL and 80 mg / mL, respectively.

[0041] In addition, Crocus sativus L. extract also showed a significant inhibitory effect on Aspergillus parasiticus (see Figure 4 、 5). Different concentrations of the extract could inhibit the growth of mycelium of A. parasiticus, and the inhibition effect was enhanced with the increase of the concentration. At the concentrations of 20 mg / mL, 40 mg / mL and 60 mg / mL, the inhibition rate gradually decreased with the extension of culture time. At the concentration of 80 mg / mL, the growth of mycelium was completely inhibited in the first 2 days, but a small amount of mycelium appeared on the third day, the colony diameter was 1.48 ± 0.08 cm, and the inhibition rate was 85.48 ± 2.99%. At the concentration of 100 mg / mL, no mycelium growth was observed in 5 days. Therefore, under the experimental conditions, the MIC and MFC of the extract of S. crocata against A. parasiticus were 80 mg / mL and 100 mg / mL, respectively.

Claims

1. A method for preparing a saffron bud flavonoid extract, characterized in that, The method specifically includes the following steps: S.

1. The dried saffron buds are crushed by a pulverizer and sieved. The powder is weighed and extracted using ultrasonic circulation extraction. The extract is filtered to obtain the supernatant. S.

2. The supernatant above is concentrated under reduced pressure to a paste-like state, and then freeze-dried to obtain the final extract.

2. The method for preparing a saffron bud flavonoid extract as described in claim 1, characterized in that, The ultrasonic cyclic extraction conditions in step S.1 are as follows: ultrasonic time is 30-50 min, ultrasonic power is 400-800 W, and stirring times are 800-1200 rpm.

3. The method for preparing a saffron bud flavonoid extract as described in claim 2, characterized in that, The temperature for ultrasonic cyclic extraction in step S.1 is 30-50℃.

4. The method for preparing a saffron bud flavonoid extract as described in claim 2, characterized in that, In step S.1, ultrasonic cyclic extraction uses an ethanol solution with a concentration of 40-80%.

5. The method for preparing a saffron bud flavonoid extract as described in claim 4, characterized in that, In step S.1, the material-to-liquid ratio for ultrasonic cyclic extraction is 20-40 mL / g of sample to ethanol solution.

6. The saffron bud flavonoid extract prepared by any one of the methods described in claims 1-5.

7. The application of the saffron bud flavonoid extract as described in claim 6 in the preparation of antibacterial products.

8. The application as described in claim 7, characterized in that, The antibacterial effect refers to the inhibition of Aspergillus flavus and Aspergillus parasiticus.

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