Method for extracting coumarin components from oldham centella

Abstract

The application discloses a method for extracting coumarin components from old mountain celery, and belongs to the technical field of natural plant effective component extraction. The steps of the extraction method comprise the following steps: using an ethanol aqueous solution containing alkyl glycoside surfactants as an extraction liquid, and extracting coumarin from the old mountain celery through ultrasonic extraction. The application selects the ethanol aqueous solution as a main extraction agent, adds alkyl glycoside surfactants with good biodegradability, achieves the effect of improving the extraction rate of the coumarin extracted from the old mountain celery, and further combines the ultrasonic extraction method, so that the coumarin can be maximally extracted from the old mountain celery.

Description

Technical Field

[0001] This invention belongs to the field of natural plant active ingredient extraction technology, specifically relating to a method for extracting coumarin components from *Pheretima aspergillum*. Background Technology

[0002] Old Mountain Celery (Heracleum moellendorffii Hance, HM), also known as Northeast Bull Angelica, is a perennial herb belonging to the genus Heracleum in the family Apiaceae. It grows to a height of 0.7–1.5 meters, with relatively large roots that grow deep into the soil. The stem is erect, cylindrical, hollow, and ribbed, covered with spreading coarse hairs, and has a slightly distinctive aroma. The base of the stem is thickened and sheathed. Stem leaves are petiolate, with coarse hairs on the petioles. Early leaves have 3 or 5 leaflets, and the leaflets are trifoliate or pinnately compound with petiolate leaflets. In its natural state, seedlings emerge in mid-May. One-year-old leaves are elliptical. From the second year onwards, it bolts, flowers, and bears fruit. Flowering occurs from July to August, and fruiting from August to September. The flowers are white, arranged in compound umbels, and about 20 cm in diameter. The fruit is a double-schizocarp, broadly elliptical to nearly round in shape, 7–8 mm long and about 7 mm wide. *Celtis sinensis* adapts to mild, cool, and humid environments, preferring humus-rich loam and sandy loam. It has strong cold resistance, able to withstand temperatures as low as -4°C for several hours without frost damage. The underground rhizomes of mature plants can withstand -40°C without freezing. Temperature changes affect photosynthetic accumulation in *Celtis sinensis*, and are one of the important factors influencing its growth. Studies have shown that the optimal average daily temperature for *Celtis sinensis* growth is 20–25°C.

[0003] Wild celery, a common edible and medicinal vegetable in Northeast China, is beloved for its unique taste and rich nutritional value. Its main chemical components include coumarins, polyphenols, flavonoids, polysaccharides, and other active ingredients.

[0004] Coumarins are a class of lactone compounds widely distributed in nature, possessing rich biological activities due to their diverse chemical structures. As a result, coumarins are widely used in pharmaceuticals, daily chemicals, and agrochemicals. In practical production, coumarins are used as antifungal agents, natural antioxidants, and anticoagulants. Furthermore, as important secondary metabolites in plants, coumarins are crucial to plant protection mechanisms, helping plants resist external infections. In addition, in plant physiology, coumarins can also act as antioxidants, enzyme inhibitors, and precursors to toxic substances. More notably, some coumarins can also affect plant growth, such as influencing the activity of growth hormones and regulators, and can also be important factors affecting plant respiration and photosynthesis.

[0005] Traditional methods for extracting coumarins include ultrasonic extraction, microwave extraction, and hot reflux extraction, but their extraction efficiency is low. Currently, there are studies on using surfactants in combination with other traditional extraction methods to enhance coumarin extraction efficiency, but no studies have been specifically conducted on *Pheretima aspergillum*. Given the complexity of natural plant components, further research is needed to determine which surfactants, combined with which traditional extraction methods, can improve the extraction rate of coumarins from *Pheretima aspergillum*. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting coumarin components from wild celery. By using alkyl glycosides as surfactants, combined with an ethanol-water solution and ultrasonic extraction, coumarins can be extracted from wild celery to the maximum extent.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for extracting coumarin components from wild celery includes the following steps:

[0009] Coumarin was extracted from *Pheretima aspergillum* using an ethanol-water solution containing alkyl glycoside surfactants as the extraction solvent and by ultrasonic extraction.

[0010] Preferably, the alkyl glycoside surfactant is octylquinoline.

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

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

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

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

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

[0016] This invention selects an aqueous ethanol solution as the main extraction agent, and by adding alkyl glycoside surfactants with good biodegradability, the extraction rate of coumarin from celery is improved. Furthermore, by combining ultrasonic extraction, coumarin can be extracted from celery to the maximum extent. Attached Figure Description

[0017] Figure 1 The graph shows the effect of different surfactant concentrations on the coumarin extraction efficiency in Example 2.

[0018] Figure 2 The graph shows the effect of different ethanol concentrations on the coumarin extraction efficiency in Example 2.

[0019] Figure 3 The graph shows the effect of different material-to-liquid ratios on the coumarin extraction efficiency in Example 2.

[0020] Figure 4 The graph shows the effect of different ultrasound times on the coumarin extraction efficiency in Example 2.

[0021] Figure 5 This is a response surface plot showing the pairwise interactions of liquid-to-solid ratio, ethanol volume fraction, and surfactant concentration in Example 2. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0023] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are 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 octylquinoline glucoside (APG0810) were selected and prepared into 0.012 mg / mL surfactant solutions using 50% ethanol, pure water, ethanol, acetone, and ethyl acetate, respectively. The original solvent was used as a blank control. The absorption spectra of each surfactant solution were obtained by scanning in the wavelength range of 190-600 nm. Surfactants that did not interfere with the determination results were selected for further experiments. Dried wild celery was pulverized to 60 mesh. 0.5 g of wild celery powder was weighed and extracted at a material-to-liquid ratio of 1:40 at an ultrasonic temperature of 50℃ and an ultrasonic power of 300 W for 60 min. After centrifugation for 10 min, the supernatant was collected. This process was repeated three times. The total coumarin content in the collected supernatant was determined by ultraviolet spectrophotometry using imperatorin as a standard. Each experiment was performed three times independently. The test results are shown in Table 1.

[0029] Table 1 Extraction effects of different extraction solvents

[0030] Solvent types 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] Table 1 shows that the addition of surfactants improved the extraction efficiency of the solvent to some extent. In terms of solvent type, the extraction effect obtained using a 50% ethanol aqueous solution was better than that obtained using pure water and ethanol. This may be because the mixed solution of water and ethanol can dissolve both the hydrophilic and hydrophobic portions of coumarin during extraction, thus better extracting coumarin from the raw material. Meanwhile, APG0810 showed the most significant improvement in extraction efficiency among all surfactants. This may be because alkyl glycoside compounds containing complex carbon atoms more easily reduce the solid-liquid interfacial tension, increasing the solubility and exudation capacity of organic matter, thereby improving the extraction rate. Table 1 shows that the APG0810 solvent prepared with ethanol aqueous solution is the optimal extraction solvent for coumarin extraction from *Pheretima aspergillum*. Further optimization of the content of each component in the extraction solvent, extraction methods, and extraction parameters will be conducted subsequently.

[0032] Example 2

[0033] Extraction method optimization:

[0034] Coumarin was extracted from *Pheretima aspergillum* using 50 vol.% ethanol aqueous solution with APG0810 concentrations of 0.006, 0.008, 0.010, 0.012, and 0.014 mg / mL as the extraction solvent, under the following conditions: solid-liquid ratio 1 g: 60 mL, ultrasonic power 300 W, ultrasonic time 60 min, and extraction temperature 50 °C. The extraction results are shown in the figure. Figure 1 ( Figure 1There are significant differences in the results represented by different letters.

[0035] Figure 1 The results showed that the extraction rates of total coumarins and the three main substances in *Pheretima aspergillum* exhibited the same trend: within the range of 0.006-0.01 mg / mL, the extraction rates increased with increasing APG0810 concentration, and then gradually decreased with further increases in concentration. This may be due to the unique structural properties of surfactants: below the critical micelle concentration (CMC), surfactants exist as dispersed rod-like structures in solution and cannot form stable micelle structures, thus some active substances are oxidized and degraded during extraction. Conversely, when the surfactant concentration is too high, its viscosity also increases, leading to a decrease in extraction efficiency. The highest extraction rate was achieved at a concentration of 0.010 mg / mL, with extraction rates of 1.23 mg / g, 6.65 mg / g, 7.66 mg / g, and 1.63 mg / g for anethole, imperatorin, saxifragein, and bergamot lactone, respectively. Total coumarins also achieved optimal extraction. Therefore, the optimal surfactant concentration was selected as 0.01 mg / mL.

[0036] Coumarin was extracted from *Pheretima aspergillum* using 40 vol.%, 50 vol.%, 60 vol.%, 70 vol.%, and 80 vol.% ethanol aqueous solutions with an APG0810 content of 0.010 mg / mL as extraction solvents, under the conditions of 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 the figure. Figure 2 ( Figure 2 There are significant differences in the results represented by different letters.

[0037] Figure 2 The results showed that within the ethanol volume fraction range of 40%-80%, the three coumarins in *Pheretima aspergillum* exhibited a trend of first increasing and then decreasing. This may be related to the properties of alkyl glycoside surfactants; the solubility of alkyl glycosides in water decreases with increasing APG carbon chain length. Increasing the ethanol volume fraction can promote the solubility of APG0810 and increase the extraction rate. However, continuously increasing the ethanol concentration can lead to the dissolution of some fat-soluble components in *Pheretima aspergillum*, affecting the extraction effect. During the extraction of coumarins from *Pheretima aspergillum*, the optimal extraction effects for anethole lactone, imperatorin, saxifragein, and bergamot lactone were achieved at an ethanol volume fraction of 60%, with concentrations of 1.12 mg / g, 5.45 mg / g, 6.65 mg / g, and 1.67 mg / g, respectively.

[0038] Coumarin was extracted from *Pheretima aspergillum* using a 60 vol.% ethanol aqueous solution with an APG0810 content of 0.010 mg / mL as the extraction solvent, under the following conditions: solid-liquid ratios of 1 g:20 mL, 1 g:40 mL, 1 g:60 mL, 1 g:80 mL, and 1 g:100 mL; ultrasonic power of 300 W; ultrasonic time of 60 min; and extraction temperature of 50 °C. The extraction results are shown in the figure. Figure 3 ( Figure 3 There are significant differences in the results represented by different letters.

[0039] Figure 3 The results showed that the extraction rate of coumarin varied greatly with different material-to-liquid ratios, with the optimal extraction effect achieved at 80 mL / g. Increasing the solution volume further at this point would lead to a decrease in the extraction rate.

[0040] Coumarin was extracted from *Pheretima aspergillum* using a 60 vol.% ethanol aqueous solution with an APG0810 content of 0.010 mg / mL as the extraction solvent, under the following conditions: a solid-liquid ratio of 1 g:60 mL, an ultrasonic power of 300 W, ultrasonic times of 20, 40, 60, 80, and 100 min, and an extraction temperature of 50 °C. The extraction results are shown in the figure. Figure 4 ( Figure 4 There are significant differences in the results represented by different letters.

[0041] Figure 4 The results showed that when the ultrasonic time was within 10-60 min, the extraction rate of coumarin substances increased with the extension of ultrasonic time, and the extraction rate reached the maximum at 60 min. The extraction rates of anethole, imperatorin, saxifragein and bergamot lactone were 1.02 mg / g, 6.02 mg / g, 7.38 mg / g and 1.53 mg / g, respectively.

[0042] Optimal extraction parameters were further determined using response surface methodology: Box-Behnken design (BBD) was employed in conjunction with response surface methodology (RSM) to explore optimal extraction conditions. Based on the results of single-factor experiments, several key factors were identified as the 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. A three-factor, three-level experiment was conducted using 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 using Design-Expert 10.0.7 software, yielding the polynomial regression model equation Y = 25.43 + 0.70A - 0.456B - 0.72C + 0.57AB + 0.30AC - 1.02BC - 3.09A² - 1.14B 2 -4.25C 2 An analysis of variance was performed on the model, and the results are shown in Table 3.

[0047] Table 3. Analysis of Variance for Response Surface Models

[0048] Sources of difference sum of squares Degrees of freedom Mean squared deviation F value 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 Misfit Error 0.21 3 0.070 1.38 0.3711 Pure error 0.20 4 0.051 sum 148.32 16

[0049] The model's F = 279.49 and P < 0.0001 indicate that the model is significant. The P-value lacking fit term > 0.05 and the F-value lacking fit term > 0.5 indicate a good model fit. R² = 0.9972, coefficient of variation (CV) = 1.13%, and R²adj = 0.9753 indicate good model correlation. In the first-order terms, A and C have significant effects; in the interaction terms, BC has a significant effect; and in the second-order terms, A... 2 B 2 C2 has 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 plots of the pairwise interactions between liquid-to-solid ratio, ethanol volume fraction, and surfactant concentration are shown in [reference needed]. Figure 5 ,Depend on Figure 5It can be seen that the steep slope of the response surface, resembling a bell jar, indicates that the interaction between factors B and C has a significant impact on the extraction of apigenin. The relatively gentle slope indicates that the interactions 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 plot are consistent with the variance analysis results in Table 3-6. Therefore, this model can be used to determine the optimal extraction conditions for surfactant-assisted ultrasonic extraction of apigenin. Analysis using Design-expert 10.0.7 software revealed the optimal extraction conditions for coumarin to be a liquid-to-solid ratio of 81.92 mL / g, an ethanol volume fraction of 58.49%, and a surfactant concentration of 0.0098 mg / mL, predicting an extraction rate of 20.02 mg / g. Considering 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. Extraction was carried out under the modified conditions, and the extraction rates of bergamot lactone, imperatorin, anethole, 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 total coumarin extraction rate was 19.83 mg / g, which was close to the response surface methodology prediction, proving that the model is reasonable and effective.

[0051] Investigation of surfactant combined with traditional extraction methods: Coumarin was extracted from *Pheretima aspergillum* 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 under optimal conditions for all methods, SUE extraction achieved the best efficiency, with a significantly higher extraction rate of coumarin from *Pheretima aspergillum* compared to other methods. Compared to surfactant-assisted water bath extraction (SWE), this method is faster, requires lower temperatures, and has a 17.65% higher efficiency. Compared to ultrasonic extraction (UAE), the addition of surfactants significantly increases the coumarin extraction rate from *Pheretima aspergillum* and reduces the extraction time. This indicates that surfactant-assisted ultrasonic extraction is a highly efficient extraction technique.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting coumarin components from *Pheretima aspergillum*, characterized in that, Includes the following steps: Coumarin was extracted from *Pheretima aspergillum* by ultrasonic extraction using an ethanol-water solution containing alkyl glycoside surfactants. The alkyl glycoside surfactant is octyldecyl glucoside; The volume fraction of ethanol in the ethanol-water solution is 58%. The content of alkyl glycoside surfactant in the ethanol aqueous solution is 0.01 mg / mL; The material-to-liquid ratio during extraction was 1g:82mL; The ultrasonic extraction was performed at a power of 300W, a time of 60 minutes, and a temperature of 50℃.

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