Method for extracting total flavonoids from scabrous doellingeria rhizome and root stems

Abstract

The invention belongs to the technical field of natural medicine extraction, and particularly relates to a method for extracting total flavonoids from scabrous doellingeria rhizome and root stems, which comprises the following steps: S1, cleaning fresh scabrous doellingeria rhizome and root stems, drying to constant weight, crushing and sieving; s2, soaking scabrous doellingeria rhizome powder in an ethanol solution for 30 minutes; performing reflux extraction in a water bath kettle, cooling to room temperature, and filtering to obtain a flavone crude extract; s3, adding petroleum ether into the crude flavone extracting solution, and extracting with an extracting agent to obtain an extracting solution; and S4, concentrating and drying the obtained extract liquor to obtain the scabrous doellingeria rhizome and root total flavonoids. The extraction method disclosed by the invention is simple and efficient in process and high in total flavone extraction yield, and can provide powerful support for development of main active ingredients of scabrous doellingeria rhizome and root medicinal materials. In addition, experiments find that the scabrous doellingeria rhizome total flavonoids have a relatively strong inhibition effect on escherichia coli and also have an inhibition effect on staphylococcus aureus.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine extraction, and particularly relates to a method for extracting total flavonoids from bougainvillea stems. Background Art

[0002] Dongfengcai (Asteraceae) is a herbaceous plant in the Asteraceae family. It is mentioned in ancient Chinese medical texts. It is not only a refreshing wild vegetable, but also boosts immunity, promoting health and longevity. It has the effects of clearing away heat and detoxifying, dispelling wind and relieving pain. It is used to treat snake venom, enteritis, typhoid fever, pharyngitis, and arthritis, and has rich medicinal value. Dongfengcai contains a variety of chemical components, primarily terpenes, flavonoids, saponins, steroids, and sugars. In recent years, researchers at home and abroad have published numerous reports on the active ingredient total saponins in Dongfengcai roots, but fewer studies have examined the active ingredients total flavonoids in Dongfengcai stems and leaves.

[0003] Flavonoids are a type of polybasic metabolite that normally appears as yellow or light yellow crystalline powders, with a few being non-crystalline amorphous powders. They have a wide range of physiological activities and low toxicity. Studies have shown that flavonoids have anti-free radical, cancer prevention, and tumor cell apoptosis-inducing effects. They also show good effects in preventing diabetes and kidney disease, and have good therapeutic effects in antibacterial, antiviral, immune-enhancing, liver-protecting, and lipid-lowering aspects. Through a search of the prior art, current research on total flavonoids in the stems and leaves of Ailanthus altissima has mainly focused on their efficacy and effects, while there are few reports on the extraction of total flavonoids from the stems and leaves of Ailanthus altissima. Therefore, providing a method for extracting total flavonoids from the stems of Ailanthus altissima is of great significance for the development of total flavonoids, the main active ingredient in Ailanthus altissima medicinal materials. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting total flavonoids from the stems of Dongfengcai, in order to provide strong support for the development of total flavonoids, the main active ingredient in Dongfengcai medicinal materials.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for extracting total flavonoids from celery stems comprises the following steps: S1. Raw material processing Remove the leaves and roots of fresh Dongfengcai, leaving the stems, wash and air dry, dry to constant weight, crush and sieve; S2. Extraction Take Dongfengcai stem powder, add ethanol solution at a solid-liquid ratio of 1g:10-30mL, and soak for 30 minutes; reflux extraction in a 50-90℃ water bath, cool to room temperature, filter with gauze, and collect the filtrate to obtain the crude flavonoid extract; S3. Extraction Add petroleum ether to the crude flavonoid extract, and add an extractant at the same time, remove the lower layer after extraction, and repeat twice; then extract with an equal amount of extractant, remove the lower layer after extraction, and repeat twice; S4. Concentration The obtained extract is concentrated to a viscous state, and then dried at 40° C. to obtain the total flavonoids from the stems of Dongfengcai.

[0006] Furthermore, in step S1, the product is dried in an oven at 40-50° C. to a constant weight and then crushed to pass through a 50-80 mesh sieve.

[0007] Furthermore, in step S2, the material-liquid ratio is 1 g:20 mL.

[0008] Furthermore, in step S2, the concentration of ethanol is 60%.

[0009] Furthermore, in step S2, the mixture is refluxed in a water bath at 70° C. for 2 h.

[0010] Furthermore, in step S3, the extractant is water-saturated n-butanol.

[0011] Furthermore, in step S4, the extract is placed in a rotary evaporator at a temperature of 60° C. and 40-50 rpm / min for concentration.

[0012] The present invention also provides the effect of the total flavonoids extracted from the stems of Easter twigs in inhibiting Escherichia coli and Staphylococcus aureus.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts ethanol solution as extractant, utilizes heating reflux method to extract total flavonoids from Dongfengcai stem, and uses L9 (3 3 ) The orthogonal test method was used to optimize the extraction process, and the optimal extraction process for total flavonoids from Dongfengcai stems was obtained as follows: solid-liquid ratio of 1g:20mL, ethanol concentration of 60%, extraction temperature of 70°C, and extraction time of 2h. Under these conditions, the maximum extraction rate of total flavonoids from Dongfengcai stems was 1.37%. The extraction method of the present invention is simple and efficient, with a high extraction rate of total flavonoids, which can provide strong support for the development of the main active ingredients of Dongfengcai medicinal materials. In addition, the total flavonoids from Dongfengcai stems have a strong inhibitory effect on Escherichia coli, and ... -4 mg / mL, it was statistically significant compared with the normal saline control group; at a concentration of 2.0 mg / mL, it also had an inhibitory effect on Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The standard curve is drawn with absorbance as the ordinate and rutin concentration as the abscissa; Figure 2The effect of the ratio of solid to liquid on the extraction rate of total flavonoids from Dongfengcai stems; Figure 3 The effect of ethanol concentration on the extraction rate of total flavonoids from Dongfengcai stems; Figure 4 The effect of extraction temperature on the extraction rate of total flavonoids from the stems of Dongfengcai. Figure 5 The antibacterial effect of total flavonoids from the stems of Dongfengcai on Escherichia coli (only one replicate is shown); Figure 6 The antibacterial effect of total flavonoids from the stems of Dongfengcai on Staphylococcus aureus (only one replicate is shown). DETAILED DESCRIPTION

[0015] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0016] 1. Materials and Methods 1.1 Single factor experiment and orthogonal experimental design for the extraction of total flavonoids from Dongfengcai stems Under the premise of a fixed extraction time of 2 h, the effects of the following single factors on the extraction rate of total flavonoids from Dongfengcai were investigated: a. Screen the extraction ratios at 70°C, 60% ethanol concentration, and 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL, and 1g:30mL. b. Screening of ethanol concentrations using a solid-liquid ratio of 1 g:15 mL, an extraction temperature of 70°C, and ethanol concentrations of 40%, 50%, 60%, 70%, and 80%. c. Screening temperature: 1g:15mL material-liquid ratio, 60% ethanol concentration, extraction temperatures of 50°C, 60°C, 70°C, 80°C, and 90°C; d. Based on the results of single factor experiments a, b and c, select the appropriate range of solid-liquid ratio, ethanol concentration, and extraction temperature as variables, and the dependent variable is the extraction rate of total flavonoids from Dongfengcai stems. 3 ) Orthogonal study.

[0017] 1.2 Extraction steps S1. Raw material processing Remove the leaves and roots of the collected fresh Dongfengcai, leaving the stems, wash with tap water, dry naturally in the sun, transfer to an oven to dry at 40℃ to a constant weight, grind through a 60-mesh sieve, put into a bag, seal it to prevent moisture, and set aside.

[0018] S2. Extraction Extraction a: Set the solid-liquid ratio of 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL and 1g:30mL to screen the optimal solid-liquid ratio, that is, accurately weigh 50g of the Dongfengcai stem powder of step S1, place it in a 2000mL round-bottom flask, add 500mL, 750mL, 1000mL, 1250mL and 1500mL of 60% ethanol solution, respectively, and soak for 30min; heat the water bath temperature to 70°C, place the reflux device in the water bath, hot reflux extraction for 2h, then cool to room temperature, filter the filtrate with gauze to obtain the crude flavonoid extract.

[0019] Extraction b: Set the material-liquid ratio to 1g:15mL, the extraction temperature to 70°C, and the ethanol concentration to 40%, 50%, 60%, 70% and 80%, and screen the optimal ethanol concentration. That is, accurately weigh 50g of the Dongfengcai stem powder from step S1, place it in a 1000mL round-bottom flask, add 750mL of ethanol, and the ethanol concentrations are 40%, 50%, 60%, 70%, and 80%, respectively, and soak for 30 minutes; heat the water bath temperature to 70°C, place the reflux device in the water bath, and perform hot reflux extraction for 2 hours, then cool to room temperature, and filter the filtrate with gauze to obtain the crude flavonoid extract.

[0020] Extraction c: Set the material-liquid ratio to 1g:15mL, the ethanol concentration to 60%, the extraction temperatures to 50℃, 60℃, 70℃, 80℃ and 90℃, and screen the optimal extraction temperature. That is, accurately weigh 50g of the above-mentioned crushed Dongfengcai stem powder, place it in a 1000mL round-bottom flask, add 750mL of 60% concentration ethanol solution, and soak for 30min; heat the water bath to the corresponding temperatures (50℃, 60℃, 70℃, 80℃, 90℃), place the reflux device in the water bath, and perform hot reflux extraction for 2h, then cool to room temperature, and filter the filtrate with gauze to obtain the crude flavonoid extract.

[0021] S3. Extraction The crude flavonoid extract of S2 was placed in a separating funnel, and an equal amount of petroleum ether was added. At the same time, an equal volume of water-saturated n-butanol was added to extract for 30 min, the lower layer was removed, and the process was repeated twice. Then, an equal amount of water-saturated n-butanol was used to extract for 30 min, the lower layer was removed, and the process was repeated twice to obtain the extract.

[0022] S4. Concentration The extract of S3 was concentrated in a rotary evaporator at a temperature of 60°C and a rotation speed of 45 rpm / min until it became viscous. The extract was then poured into a Petri dish and dried in an oven at 60°C to a constant weight. The total flavonoids of the stem of Dongfengcai were obtained by weighing.

[0023] S5. Calculation of the total flavonoid extraction rate from Dongfengcai stems Accurately weigh 10 mg of rutin standard, dissolve it in 60% ethanol solution, transfer it to a 100 mL volumetric flask and dilute to the scale to obtain a rutin standard solution with a mass concentration of 0.1 mg / mL. Accurately aspirate 0, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, and 3.50 mL of rutin standard solution, respectively, and add them to the marked 10 mL test tubes in turn. Add 0.3 mL of 5% NaNO2 solution to each test tube in turn, mix well, let it stand for 6 minutes, then add 0.30 mL of 10% Al(NO3)3 solution, mix well, and let it stand for 6 minutes; add 4.00 mL of 4% NaOH solution, and finally add it to 10 mL with 60% ethanol solution, mix well, let it stand for 12 minutes, and measure the absorbance at a wavelength of 510 nm. Use the concentration (C) of the rutin standard as the X-axis and the absorbance of the corresponding concentration as the Y-axis to draw a standard curve, as shown. Figure 1 The linear regression equation is Y=15.038X-0.0142, R 2 =0.996.

[0024] Take 10 mg of the dried extract and prepare a total flavonoid solution from the stem of Ailanthus truncatus according to the preparation method of the rutin standard solution. Then measure the absorbance. Substitute the measured absorbance value into the standard curve, calculate the concentration of total flavonoids, and then calculate the extraction rate of total flavonoids from Ailanthus truncatus by substituting the formula: Extraction rate of total flavonoids T (%) = , where C is the total flavonoids concentration (mg / mL), N is the dilution factor, V is the sample volume (mL), and M is the weight of Dongfengcai (mg).

[0025] According to the results of single factor experiments a, b, and c, that is, according to the total flavonoid extraction rate results of extraction a, extraction b, and extraction c, the appropriate range of solid-liquid ratio, ethanol concentration, and extraction temperature were selected as variables, and the extraction rate of total flavonoids from Dongfengcai stems was the dependent variable. L9 (3 3 ) Orthogonal experiment, the extraction results were extracted, concentrated and the yield of total flavonoids was calculated according to S3, S4 and S5 respectively.

[0026] 1.3 Antibacterial test of total flavonoids from Dongfengcai stems S1. Accurately weigh 2.0 g of the total flavonoids extracted from the stems of Dongfengcai and dissolve them in 10 mL of sterile saline to make a 200 mg / mL stock solution. Filter and sterilize. The working concentration is 2.0 mg / mL-2.0×10 -6 mg / mL; a positive control penicillin solution was prepared with a concentration of 2.0×10 -1 mg / mL.

[0027] S2. Pour the prepared LB medium (950 mL of distilled water, add 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, adjust to pH 7.0, add 15 g of agar, and distilled water to 1 L) into a 90 mm diameter Petri dish. After checking for sterility, inoculate each plate with 0.1 mL of Escherichia coli or Staphylococcus aureus suspension (both at a concentration of 1 × 10 6 cfu / mL).

[0028] S3. Use tweezers to place sterile filter paper discs into the prepared Dongfengcai stem flavonoid solutions of varying concentrations, the positive control penicillin solution, and the blank control saline solution. Allow to soak for 10 minutes. Remove the discs and place them in the corresponding areas of the culture medium plates (three filter paper discs per plate). Cover the plates with lids, setting up three replicates for each treatment. Incubate at 37°C for 2 days. Observe and measure the size of the inhibition zone using a vernier caliper.

[0029] 2. Results and Analysis 2.1 Effect of solid-liquid ratio on the extraction rate of total flavonoids from Dongfengcai stems See the results Figure 2 .

[0030] Depend on Figure 2 The yield of total flavonoids from the stems of Dongfengcai increased with increasing material-liquid ratios, reaching a maximum yield of 1.37% at a material-liquid ratio of 1g:20mL. However, the yield of total flavonoids from the stems of Dongfengcai decreased with increasing material-liquid ratios, reaching 0.91% at a material-liquid ratio of 1g:25mL and 0.93% at a material-liquid ratio of 1g:30mL, ultimately exceeding 0.95%. Considering the amount of raw materials and reagents used, material-liquid ratios of 1g:10mL, 1g:15mL, and 1g:20mL were selected for the experiments.

[0031] 2.2 Effect of ethanol concentration on the extraction rate of total flavonoids from Dongfengcai stems See the results Figure 3 .

[0032] Depend on Figure 3 The yield of total flavonoids from the stems of Easter spinach increased with increasing ethanol concentration, reaching a maximum yield of 0.89% at 60% ethanol. However, the yield of total flavonoids decreased with increasing ethanol concentration. In this study, the yield of total flavonoids was relatively high at 50%, 60%, and 70% ethanol concentrations, so these three concentrations were selected for the experiment.

[0033] 2.3 Effect of extraction temperature on the extraction rate of total flavonoids from Dongfengcai stems See the results Figure 4 .

[0034] Depend on Figure 4It can be seen that the yield of total flavonoids from the stems of Dongfengcai increases with the increase of extraction temperature. Considering the boiling point of the solvent and the biological activity of flavonoids, the extraction temperature should not be too high. Therefore, the extraction temperatures of 70℃, 80℃ and 90℃ were selected for the experiments.

[0035] 2.4 Results of orthogonal experiments According to the single factor experiment, the appropriate range of solid-liquid ratio (1g:10mL, 1g:15mL and 1g:20mL), ethanol concentration (50%, 60% and 70%), and extraction temperature (70℃, 80℃ and 90℃) were selected as variables, and the extraction rate of total flavonoids from Dongfengcai stems was the dependent variable. 3 ) orthogonal experiment (as shown in Table 1), the extraction time was 2h, and the extraction rate of total flavonoids from Dongfengcai stems was shown in Table 2.

[0036] Table 1 Orthogonal experimental factors and levels

[0037] Table 2 L9 (3 3 ) Orthogonal experimental results

[0038] Table 2 shows that the optimal extraction conditions for total flavonoids from the stems of Dongfengcai are a solid-liquid ratio of 1 g:20 mL, an ethanol concentration of 60%, and an extraction temperature of 70°C. Using these conditions, the maximum total flavonoid extraction yield was 1.37%. Furthermore, the R value indicates that the solid-liquid ratio and ethanol concentration have a significant impact on the total flavonoid extraction yield, while the extraction temperature has a relatively small effect.

[0039] 2.5 Antibacterial effect of total flavonoids from Dongfengcai stems on Escherichia coli The results are shown in Figure 5 and Table 3.

[0040] Table 3 Antibacterial effect of total flavonoids from Dongfengcai stems on Escherichia coli

[0041] Note: The antibacterial effect of total flavonoids from Dongfengcai stems on Escherichia coli was significantly different from that of the normal saline blank control group ( P <0.05), an “*” is marked in the upper right corner of the corresponding value, the diameter of the inhibition zone includes the diameter of the filter paper (6 mm), and the penicillin concentration is 2.0×10 -1 mg / mL.

[0042] Figure 5 It shows that the total flavonoids from Dongfengcai stems have a strong antibacterial effect on Escherichia coli.

[0043] The results in Table 3 show that the concentration of total flavonoids in Dongfengcai stems ranges from 2.0 mg / mL to 2.0×10 -4mg / mL showed significant difference in antibacterial effect compared with the blank control ( P<0.05 ), there is a certain dose-effect relationship between the antibacterial effect and the total flavonoids concentration of Dongfengcai stems. As the total flavonoids concentration of Dongfengcai stems decreases, the diameter of the inhibition zone gradually decreases, indicating that the antibacterial effect gradually weakens.

[0044] When the total flavonoids concentration of Dongfengcai stems was reduced to 2.0×10 -5 mg / mL-2.0×10 -6 There was no antibacterial effect when compared with the blank control. There was no significant difference in the antibacterial effect ( P >0.05). Therefore, 2.0×10 -4 mg / mL is the minimum inhibitory concentration of total flavonoids from Dongfengcai stems to Escherichia coli. When the concentration of total flavonoids from Dongfengcai stems and the concentration of penicillin are both 2.0×10 -1 mg / mL, the antibacterial effect of total flavonoids from Dongfengcai stems was not as good as that of penicillin.

[0045] 2.6 Antibacterial effect of total flavonoids from Dongfengcai stems on Staphylococcus aureus See the results Figure 6 and Table 4.

[0046] Table 4 Antibacterial effect of total flavonoids from the stems of Dongfengcai on Staphylococcus aureus

[0047] Note: The inhibitory effect of total flavonoids from Dongfengcai stem on Staphylococcus aureus was significantly different from that of the normal saline blank control group ( P <0.05), an “*” is marked in the upper right corner of the corresponding value. The diameter of the inhibition zone includes the diameter of the filter paper (6 mm). The penicillin concentration is 2.0×10 -1 mg / mL.

[0048] Figure 6 It showed that the total flavonoids in Dongfengcai stems had an antibacterial effect on Staphylococcus aureus when the concentration was 2.0 mg / mL.

[0049] The results in Table 4 show that when the total flavonoids concentration in Dongfengcai stems was 2.0 mg / mL, there was a significant difference compared with the blank control ( P <0.05), but the inhibitory effect on Staphylococcus aureus was less than 2×10 -1 mg / mL positive control penicillin solution. When the concentration of total flavonoids in Dongfengcai stems was reduced to 2×10 -1 -2×10 -2 mg / mL, there was no antibacterial effect, and there was no significant difference in antibacterial effect compared with the blank control ( P >0.05). Therefore, the minimum inhibitory concentration of total flavonoids from the stems of Dongfengcai against Staphylococcus aureus is 2.0 mg / mL.

[0050] In summary, the present invention uses ethanol solution as the extractant and utilizes the heating reflux method to extract the total flavonoids from the stems of Dongfengcai. 3 Orthogonal design was used to optimize the extraction process, revealing the optimal extraction process for total flavonoids from the stems of angelica dahurica (S. dahurica) as follows: a solid-liquid ratio of 1 g:20 mL, an ethanol concentration of 60%, an extraction temperature of 70°C, and an extraction time of 2 hours. Under these conditions, the maximum extraction yield of total flavonoids from the stems of angelica dahurica (S. dahurica) was 1.37%. This extraction method, characterized by its simplicity and efficiency, achieves a high total flavonoid yield and provides strong support for the development of the main active ingredients of angelica dahurica (S. dahurica) medicinal materials.

[0051] In addition, the total flavonoids of Dongfengcai stems had a strong inhibitory effect on Escherichia coli in the concentration range of 2.0 mg / mL-2.0×10 -4 mg / mL, it was statistically significant compared with the normal saline control group, and at a concentration of 2.0 mg / mL, it also had an inhibitory effect on Staphylococcus aureus.

Claims

1. A method for extracting total flavonoids from the stems of Oriental broccoli, characterized in that: The following steps are involved: S1. Raw material processing Remove the leaves and roots of fresh Dongfengcai, leaving the stems, wash and air dry, dry to constant weight, crush and sieve; S2. Extraction Take Dongfengcai stem powder, add ethanol solution at a solid-liquid ratio of 1g:10-30mL, and soak for 30 minutes; reflux extraction in a 50-90℃ water bath, cool to room temperature, filter with gauze, and collect the filtrate to obtain the crude flavonoid extract; S3. Extraction Add petroleum ether to the crude flavonoid extract, and add an extractant at the same time, remove the lower layer after extraction, and repeat twice; then extract with an equal amount of extractant, remove the lower layer after extraction, and repeat twice; S4. Concentration The obtained extract is concentrated to a viscous state, and then dried at 40° C. to obtain the total flavonoids from the stems of Dongfengcai.

2. The method for extracting total flavonoids from the stems of Angelica dahurica according to claim 1, characterized in that: In step S1, the product is dried in an oven at 40-50° C. to a constant weight and then pulverized to pass through a 50-80 mesh sieve.

3. The method for extracting total flavonoids from the stems of Easter peas according to claim 2, characterized in that In step S2, the material-liquid ratio is 1 g:20 mL.

4. The method for extracting total flavonoids from the stems of Easter peas according to claim 2, characterized in that In step S2, the concentration of ethanol is 60%.

5. The method for extracting total flavonoids from the stems of Easter peas according to claim 2, characterized in that In step S2, the mixture was refluxed in a water bath at 70° C. for 2 h.

6. The method for extracting total flavonoids from the stems of Easter peas according to claim 2, characterized in that In step S3, the extractant is water-saturated n-butanol.

7. The method for extracting total flavonoids from the stems of Easter spinach according to claim 2, characterized in that In step S4, the extract is placed in a rotary evaporator at a temperature of 60° C. and a speed of 40-50 rpm / min for concentration.

8. The effect of the total flavonoids extracted from the stems of Easter peas according to any one of claims 1 to 7 in inhibiting Escherichia coli and Staphylococcus aureus.

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