Method for extracting coumarin components in heracleum moellendorffii hance

Abstract

The invention discloses a method for extracting coumarin components in heracleum moellendorffii hance, and belongs to the technical field of extraction of effective components of natural plants. The extraction method comprises the following steps: by taking an ethanol water solution containing an alkyl glycoside surfactant as an extracting solution, extracting coumarin from heracleum moellendorffii through ultrasonic extraction. According to the present invention, the ethanol aqueous solution is adopted as the main extraction agent, the alkyl glycoside surfactant with good biodegradability is added to achieve the effect of improving the extraction rate of the coumarin extracted from the heracleum moellendorffii hance, and the ultrasonic extraction method is further combined to extract the coumarin from the heracleum moellendorffii hance to the maximum extent.

Description

Technical Field

[0001] The invention belongs to the technical field of extracting effective components of natural plants, and particularly relates to a method for extracting coumarin components from celery. Background Art

[0002] Heracleum moellendorffii Hance (HM), scientifically known as Northeast Niufangfeng, also known as short-haired Duhuo, is a perennial herb belonging to the genus Duhuo of the Apiaceae family. Heracleum moellendorffii Hance is a perennial herb of the Apiaceae family, with a plant height of about 0.7 to 1.5 meters, relatively thick roots and deep growth in the soil, upright, cylindrical, hollow stems, with ribs on the surface, and coarse hairs, with a slight special aroma. The base is hypertrophic and sheath-like, and the stem leaves have petioles, which are coarsely hairy. The early petioles have 3 or 5 leaflets, and the leaves are three or pinnately compound leaves with petioles. Under natural growth conditions, the seedlings of Heracleum moellendorffii Hance emerge in mid-to-early May. The leaves of the one-year-old Heracleum moellendorffii are elliptical, and they begin to bolt, bloom and bear fruit in the second year. The flowering period of Heracleum moellendorffii is from July to August, and the fruiting period is from August to September. The flowers are white and compound umbrella-shaped, with a diameter of about 20 cm. The fruit is a double hanging fruit, with a wide oval or even nearly round shape, 7 to 8 mm long and about 7 mm wide. The old mountain celery adapts to mild, cool, and humid environmental conditions. It likes loam and sandy loam with a lot of humus. It has strong cold resistance and can survive for several hours at -4°C without freezing. The underground rhizomes of mature plants can withstand -40°C without freezing to death. Temperature changes will affect the photosynthetic accumulation of the old mountain celery, which is one of the important factors affecting the growth of the old mountain celery. According to research by scholars, the most suitable daily average temperature for the growth of the old mountain celery is 20 to 25°C.

[0003] As a wild vegetable commonly used in Northeast China for both medicinal and edible purposes, old mountain celery is loved by people for its unique taste and rich nutritional value. The main chemical components of old mountain celery include coumarin, polyphenols, flavonoids, polysaccharides and other active ingredients.

[0004] Coumarin compounds are a class of lactone compounds that are widely distributed in nature and have rich biological activities due to their diverse chemical structures. For this reason, coumarin compounds have been widely used in many fields such as pharmaceuticals, daily chemicals, and agricultural chemicals. In actual production, coumarin substances are used as antifungal drugs, natural antioxidants, anticoagulants, etc. In addition, as an important secondary metabolite in plants, coumarin is crucial to the protective mechanism of plants and helps plants resist external infections. In addition, in terms of plant physiology, coumarin can also be used as antioxidants, enzyme inhibitors, and precursors of toxic substances. More noteworthy is that some coumarins can also affect plant growth, such as affecting the activity of growth hormones and regulators, and can also be used as important factors affecting plant respiration and photosynthesis.

[0005] Traditional coumarin extraction methods include ultrasonic extraction, microwave extraction and hot reflux extraction, but the extraction efficiency is not high. There are also related studies on the use of surfactants combined with other traditional extraction methods to enhance the extraction efficiency of coumarin, but there is no research on the extraction of celery. The natural plant components are complex, and it remains to be studied what kind of surfactant to choose with what traditional extraction method to improve the extraction rate of coumarin in celery. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting coumarin components from celery. By using alkyl glycoside compounds as surfactants, ethanol aqueous solution, and ultrasonic extraction, coumarin can be extracted from celery to the maximum extent.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for extracting coumarin components from old celery comprises the following steps:

[0009] Coumarin was extracted from Rhizoma Cibotii by ultrasonic extraction using ethanol aqueous solution containing alkyl glycoside surfactant as the extracting solution.

[0010] Preferably, the alkyl glycoside surfactant is octyl glucoside.

[0011] Preferably, the volume fraction of ethanol in the ethanol aqueous solution is 58%.

[0012] Preferably, the content of the alkyl glycoside surfactant in the ethanol aqueous solution is 0.01 mg / mL.

[0013] Preferably, the solid-liquid ratio during extraction is 1 g:82 mL.

[0014] Preferably, the power of the ultrasonic extraction is 300 W, the time is 60 min, and the temperature is 50°C.

[0015] The beneficial technical effects of the present invention are as follows:

[0016] The present invention selects ethanol aqueous solution as the main extractant, and achieves the effect of improving the extraction rate of coumarin from the old celery by adding an alkyl glycoside surfactant with good biodegradability, and further combines the ultrasonic extraction method to extract coumarin from the old celery to the maximum extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of different surfactant concentrations on the extraction efficiency of coumarin in Example 2.

[0018] Figure 2 This is a graph showing the effect of different ethanol concentrations on the extraction efficiency of coumarin in Example 2.

[0019] Figure 3 This is a diagram showing the effect of different material-liquid ratios on the extraction efficiency of coumarin in Example 2.

[0020] Figure 4 This is a graph showing the effect of different ultrasonic times on the extraction efficiency of coumarin in Example 2.

[0021] Figure 5 This is the response surface diagram of the pairwise interaction between liquid-to-solid ratio, ethanol volume fraction and surfactant concentration in Example 2. DETAILED DESCRIPTION

[0022] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0023] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Example 1

[0027] Selection of surfactants for extraction:

[0028] Surfactants sodium dodecyl sulfate (SDS), trimethylammonium bromide (CTAB), polyethylene glycol, Tween-80, tea saponin and octyl glucoside (APG0810) were selected, and 0.012 mg / mL surfactant solutions were prepared with 50% ethanol, pure water, ethanol, acetone and ethyl acetate, respectively. The original solvent was used as a blank control, and the absorption spectrum of each surfactant solution was obtained by scanning in the wavelength range of 190-600nm. The surfactant that does not interfere with the measurement results was selected for the next experiment. The dried old mountain celery was crushed into 60 meshes, and 0.5g of old mountain celery powder was weighed. It was extracted for 60 minutes at a material-liquid ratio of 1:40 at an ultrasonic temperature of 50℃ and an ultrasonic power of 300W. The supernatant was collected after centrifugation for 10 minutes, and repeated three times. The collected supernatant was used as a standard to detect the total coumarin content in the solution by ultraviolet spectrophotometry. Each group of experiments was repeated three times independently. The test results are shown in Table 1.

[0029] Table 1 Extraction effects of different extraction solvents

[0030] Solvent type Total coumarin extraction rate (mg / g) Ethanol 10.54±0.72f water 8.09±1.55i 50% ethanol 11.02±0.77d Ethyl acetate 10.21±1.58f acetone 9.89±1.34h APG0810+50% ethanol 15.51±0.42a APG0810+water 11.12±1.46d Tween-80 + 50% ethanol 13.31±0.33b Tween-80+water 10.42±0.92g Polyethylene glycol + 50% ethanol 10.68±1.01e Polyethylene glycol + water 9.97±0.47h SDS + 50% ethanol 11.57±0.85c CTAB + 50% ethanol 12.83±1.12b

[0031] It can be seen from Table 1 that the addition of surfactants improves the extraction effect of the solvent to a certain extent. From the perspective of the type of solvent, the extraction effect obtained by using 50% ethanol aqueous solution as a solvent is better than that of pure water and ethanol. This may be because the mixed solution of water and ethanol can dissolve both the hydrophilic part and the hydrophobic part of coumarin during extraction, so it can better extract coumarin from the raw material. At the same time, the effect of APG0810 on the improvement of the extraction effect is the most obvious among all surfactants. This may be because the alkyl glycoside compounds containing complex carbon are more likely to reduce the solid-liquid interfacial tension, increase the dissolution and exudation ability of organic matter, and thus improve the extraction rate. Table 1 shows that the APG0810 solvent prepared with ethanol aqueous solution is the best extraction solvent for extracting coumarin from old mountain celery. The content of each component in the extraction solvent, the extraction method and the extraction parameters will be further optimized in the future.

[0032] Example 2

[0033] Extraction method optimization:

[0034] Using 50 vol.% ethanol aqueous solution with APG0810 content of 0.006, 0.008, 0.010, 0.012, and 0.014 mg / mL as the extractant, coumarin was extracted from old celery at a solid-liquid ratio of 1 g:60 mL, ultrasonic power of 300 W, ultrasonic time of 60 min, and extraction temperature of 50 ° C. The extraction results are shown in Figure 1 ( Figure 1Different letters indicate significant differences among the results).

[0035] Figure 1 The results showed that the extraction rate of total coumarins and the extraction rate of the three main substances in old celery had the same trend. In the range of 0.006-0.01 mg / mL, they all increased with the increase of APG0810 concentration, and then the extraction rate gradually decreased with the increase of concentration. The reason may be the unique structural properties of surfactants: below the critical micelle concentration (CMC), the surfactant is a dispersed rod-like structure in the solution and cannot form a stable micelle structure. Therefore, some active substances will be oxidized and degraded during the extraction process. When the concentration of surfactant is too high, its viscosity will also increase, resulting in a decrease in the extraction effect. When the concentration is 0.010 mg / mL, the extraction rate is the highest. The extraction rates of anise lactone, imperatorin, saxifragein and bergamot lactone are 1.23 mg / g, 6.65 mg / g, 7.66 mg / g and 1.63 mg / g, respectively. The total coumarin also achieved the best extraction effect. Therefore, the optimal concentration of surfactant was selected as 0.01 mg / mL.

[0036] Coumarin was extracted from the old celery with 40vol.%, 50vol.%, 60vol.%, 70vol.%, and 80vol.% ethanol aqueous solutions with APG0810 content of 0.010mg / mL as extractants at a solid-liquid ratio of 1g:60mL, ultrasonic power of 300W, ultrasonic time of 60min, and extraction temperature of 50℃. The extraction results are shown in Figure 2 ( Figure 2 Different letters indicate significant differences among the results).

[0037] Figure 2 It shows that within the range of 40%-80% ethanol volume fraction, the three coumarins in old celery show a trend of first increasing and then decreasing, which may be related to the properties of alkyl glycoside surfactants. The solubility of alkyl glycosides in water deteriorates with the increase of APG carbon chain. Increasing the volume fraction of ethanol can promote the solubility of APG0810 and increase the extraction rate. However, the continuous increase in ethanol concentration will cause some fat-soluble components in old celery to dissolve, affecting the extraction effect. In the extraction process of coumarin substances in old celery, when the volume fraction of ethanol is 60%, the extraction effects of anise lactone, imperatorin, saxifragein and bergamot lactone all reach the best, which are 1.12mg / g, 5.45mg / g, 6.65mg / g and 1.67mg / g respectively.

[0038] Using 60 vol.% ethanol aqueous solution with APG0810 content of 0.010 mg / mL as the extractant, coumarin was extracted from old celery at the conditions of solid-liquid ratio of 1g:20mL, 1g:40mL, 1g:60mL, 1g:80mL, 1g:100mL, ultrasonic power of 300W, ultrasonic time of 60min, and extraction temperature of 50℃. The extraction results are shown in Figure 3 ( Figure 3 Different letters indicate significant differences among the results).

[0039] Figure 3 It was shown that the extraction rate of coumarin varied greatly with the different solid-liquid ratios, and the best extraction effect was achieved at 80 mL / g. Further increasing the solution volume at this time would lead to a decrease in the extraction rate.

[0040] Coumarin was extracted from the old celery with 60 vol.% ethanol aqueous solution containing 0.010 mg / mL of APG0810 as the extractant at a solid-liquid ratio of 1 g:60 mL, an ultrasonic power of 300 W, ultrasonic times of 20, 40, 60, 80, 100 min, and an extraction temperature of 50 °C. The extraction results are shown in Figure 4 ( Figure 4 Different letters indicate significant differences among the results).

[0041] Figure 4 It shows that when the ultrasonic time is within 10-60min, the extraction rate of coumarin substances increases with the extension of ultrasonic time, and the extraction rate reaches the maximum at 60min. The extraction rates of anise lactone, imperatorin, saxifragein and bergamot lactone are 1.02mg / g, 6.02mg / g, 7.38mg / g and 1.53mg / g, respectively.

[0042] The optimal extraction parameters were further determined by response surface methodology: The Box-Behnken design (BBD) was designed and combined with the response surface method (RSM) to explore the optimal extraction conditions. Based on the results of the single factor experiment, several main factors were screened out, namely, liquid-to-solid ratio (A, ml / g), ethanol volume fraction (B, vol.%), and surfactant concentration (C, mg / mL), with the total coumarin extraction rate (Y, mg / g) as the response value. The three-factor three-level experiment was completed by BBD, and the BBD experimental results are detailed in Table 2.

[0043] Table 2 BBD experimental results

[0044]

[0045]

[0046] The experimental results were fitted by multivariate fitting using Design-Expert 10.0.7 software, and the polynomial regression model equation was obtained as Y = 25.43 + 0.70A - 0.456B - 0.72C + 0.57AB + 0.30AC - 1.02BC - 3.09A2 - 1.14B 2 -4.25C 2 , the model was subjected to variance analysis, and the results are shown in Table 3.

[0047] Table 3 Analysis of variance of response surface model

[0048] Sources of Difference sum of squares Degrees of Freedom Mean square error F-number P-value Significance Model 147.91 9 16.43 279.49 <0.0001 ** A 3.94 1 3.94 67.05 <0.0001 ** B 1.69 1 1.69 28.70 0.0011 ** C 4.12 1 4.12 70.05 <0.0001 ** AB 1.29 1 1.29 21.93 0.0023 * AC 0.35 1 0.35 5.98 0.0444 * BC 4.16 1 4.16 70.72 <0.0001 ** <![CDATA[A 2 ]]> 40.20 1 40.20 683.70 <0.0001 ** <![CDATA[B 2 ]]> 5.45 1 5.45 92.61 <0.0001 ** <![CDATA[C 2 ]]> 75.90 1 75.90 1290.72 <0.0001 ** error 0.41 7 0.059 Lack of Fit Error 0.21 3 0.070 1.38 0.3711 Pure Error 0.20 4 0.051 sum 148.32 16

[0049] The F of the model is 279.49 and P < 0.0001, indicating that the model is significant. P lack of fit > 0.05, F lack of fit > 0.5, indicating that the model fit is good. R2 = 0.9972, coefficient of variation (CV) = 1.13%, R2adj = 0.9753, indicating that the model is well correlated. The simulated linear terms A and C have significant effects, the interaction term BC has significant effects, AC and AB have no significant effects, and the quadratic term A has no significant effects. 2 , B 2 , C2 have a significant impact, and the order of influence of the three factors on the model is: liquid-to-solid ratio (A)> surfactant concentration (B)> ethanol volume fraction (C).

[0050] The response surface diagram of the pairwise interaction between liquid-to-solid ratio, ethanol volume fraction and surfactant concentration is shown in Figure 5 ,Depend on Figure 5It can be seen that the slope of the response surface is steeper and bell-shaped, indicating that the interaction between factor B and factor C has a significant effect on the extraction of coumarin from old celery. The surface is relatively flat, indicating that the interaction between factors A and C, and factors A and B have no significant effect on the response value. The above analysis results of the response surface diagram are consistent with the variance analysis results in Table 3-6, so the model can be used to determine the optimal extraction process conditions for surfactant-assisted ultrasonic extraction of coumarin from old celery. Through the analysis of Design-expert10.0.7 software, the optimal extraction conditions for coumarin are liquid-to-solid ratio of 81.92mL / g, ethanol volume fraction of 58.49%, and surfactant concentration of 0.0098mg / mL, and the predicted coumarin extraction rate is 20.02mg / g. Taking into account the actual production needs, the process parameters were adjusted to a liquid-to-solid ratio of 82 mL / g, an ethanol volume fraction of 58%, and a surfactant concentration of 0.01 mg / mL. The extraction was carried out under the revised conditions, and the extraction rates of bergamot lactone, imperatorin, anise lactone, saxifrage, and 7-hydroxycoumarin were 1.55 mg / g, 1.68 mg / g, 7.30 mg / g, 8.21 mg / g, and 0.80 mg / g, respectively. The extraction rate of total coumarins was 19.83 mg / g, which was close to the predicted value of the response surface, proving that the model is reasonable and effective.

[0051] Investigation of surfactants combined with traditional extraction methods: Coumarin was extracted from Folium sibiricum using water bath extraction (WBE), surfactant-assisted water bath extraction (SWE), ultrasonic extraction (UAE) and surfactant-assisted ultrasonic extraction (SUE). The extraction results are shown in Table 4.

[0052] Table 4 Comparison of extraction effects of different extraction methods

[0053]

[0054]

[0055] Table 4 shows that when all methods are optimal, the SUE extraction efficiency is the best, and the extraction rate of coumarin components in celery is significantly better than other methods. Compared with surfactant-assisted water bath extraction (SWE), this method takes less time, requires lower temperature, and improves efficiency by 17.65%. Compared with ultrasonic extraction (UAE), the addition of surfactant significantly increases the extraction rate of coumarin in celery and takes less time. This shows that surfactant-assisted ultrasonic extraction technology is an efficient extraction technology.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting coumarin components from old celery, characterized in that: The following steps are involved: Coumarin was extracted from Rhizoma Cibotii by ultrasonic extraction using ethanol aqueous solution containing alkyl glycoside surfactant as the extracting solution.

2. The method for extracting coumarins from Rhizoma Cibotii according to claim 1, characterized in that: The alkyl glycoside surfactant is octyl glucoside.

3. The method for extracting coumarins from Rhizoma Cibotii according to claim 1, characterized in that: The volume fraction of ethanol in the ethanol aqueous solution is 58%.

4. The method for extracting coumarins from Rhizoma Cibotii according to claim 1, characterized in that: The content of the alkyl glycoside surfactant in the ethanol aqueous solution is 0.01 mg / mL.

5. The method for extracting coumarins from Rhizoma Cibotii according to claim 1, characterized in that: The solid-liquid ratio during extraction was 1g:82mL.

6. The method for extracting coumarins from Rhizoma Cibotii according to claim 1, characterized in that: The power of the ultrasonic extraction is 300W, the time is 60min, and the temperature is 50°C.

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