Method for green and sustainable extraction and separation of biopesticide cnidium lactone

Abstract

The invention discloses a method for green and sustainable extraction and separation of biopesticide cnidium lactone, belongs to the technical field of sustainable extraction and separation of biopesticides, and utilizes a natural eutectic solvent synthesized by xylose-levulinic acid to green extract cnidium lactone in fructus cnidii. And meanwhile, a convertible solvent heptanoic acid is utilized to enrich cnidium lactone in the natural eutectic solvent. Due to the convertibility of the heptanoic acid, the hydrophobic heptanoic acid is converted into a hydrophilic solution by adjusting the pH value, so that the hydrophobic cnidium lactone is separated and recovered from the heptanoic acid extracting solution. Thereafter, the natural eutectic solvent can be directly used for new extraction. The invention provides a novel, green and sustainable extraction and separation method which is successfully used for extraction and separation of biopesticide cnidium lactone.

Description

Technical Field

[0001] The invention belongs to the technical field of sustainable extraction and separation of biological pesticides, and specifically discloses a method for green and sustainable extraction and separation of the biological pesticide osthole. Background Art

[0002] Cnidium monnieri is a traditional medicinal plant of the Umbelliferae family. Cnidium monnieri contains a variety of bioactive ingredients and has a variety of medicinal activities. It is used to treat various diseases, and most of the medicinal activities are believed to be attributed to osthole. Osthole is the most abundant active compound in Cnidium monnieri. It is a coumarin derivative with multiple biological activities such as anti-inflammatory, antibacterial, anti-tumor, and anti-allergic. It can also be used as a biological pesticide with unique insecticidal and antibacterial effects. Developing a method for extracting osthole has potential application value in the development of drugs, exploration of mechanisms of action, reducing the use of chemical pesticides, protecting the ecological environment, and promoting sustainable agricultural development.

[0003] Traditional extraction methods usually use toxic solvents as extractants, dissolve cnidium monnieri in large amounts of solvents, and then obtain osthole through a time-consuming and energy-consuming solvent separation process. Summary of the invention

[0004] The present invention provides a green and sustainable method for extracting and separating the biopesticide osthole, so as to improve the technical problems of the existing osthole extraction method, such as the high toxicity of the extractant used, the large amount of which may cause environmental pollution, and the time-consuming and energy-consuming recovery of osthole.

[0005] The above-mentioned green and sustainable method for extracting and separating the biopesticide osthole comprises the following steps:

[0006] S1, Preparation of natural deep eutectic solvent

[0007] Xylose, arabinose and glucose are used as hydrogen bond donors, and levulinic acid is used as a hydrogen bond acceptor. The two components are mixed according to a preset molar ratio and heated until a uniform and transparent liquid is formed, thereby obtaining three natural low eutectic solvents of xylose-levulinic acid, arabinose-levulinic acid and glucose-levulinic acid.

[0008] S2, extraction of osthole

[0009] Accurately weigh the Cnidium monnieri powder into a centrifuge tube, add any one of xylose-levulinic acid, arabinose-levulinic acid, glucose-levulinic acid, and add a convertible solvent, perform ultrasonic simultaneous extraction and enrichment of osthole, extract the osthole into a natural low eutectic solvent phase, and concentrate it into a convertible solvent phase, then collect the convertible solvent phase, and determine the yield of osthole by high performance liquid chromatography;

[0010] S3, recovery of osthole

[0011] The osthole extract collected in step S2 is mixed with a NaOH solution to convert the solvent from hydrophobic to hydrophilic, and the osthole solid is precipitated and recovered;

[0012] S4, reuse of natural deep eutectic solvents

[0013] The natural deep eutectic solvent phase in step S2 is collected and directly used to extract new Cnidium monnieri powder.

[0014] In step S1, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1 to 1:6; and the water content of the natural deep eutectic solvent is 10 to 70%.

[0015] In step S2, the material-liquid ratio of Cnidium monnieri powder to natural deep eutectic solvent is 1:5-1:20 g mL -1 .

[0016] In step S2, the solvent can be converted into any one of hexanoic acid, heptanoic acid, octanoic acid, and nonanoic acid.

[0017] In step S2, the volume ratio of the natural deep eutectic solvent to the convertible solvent is 10:1 to 5:3.

[0018] In step S2, the ultrasonic temperature is 20-60°C, the ultrasonic frequency is 40 Hz, and the ultrasonic time is 10-50 min.

[0019] Preferably, in step S1, the hydrogen bond donor is xylose, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:4, and the water content of the natural deep eutectic solvent is 50%;

[0020] In step S2, the material-liquid ratio of Cnidium monnieri powder to natural deep eutectic solvent is 1:15 g mL -1 , the convertible solvent is heptanoic acid, the volume ratio of the natural low eutectic solvent to the convertible solvent is 5:1, the ultrasonic temperature is 30°C, and the ultrasonic time is 30 min.

[0021] In step S2, the yield of osthole was detected by Agilent 1260 high performance liquid chromatography-diode array, and the detection conditions were: injection volume of 20 μL, mobile phase of methanol and water, the volume ratio of methanol to water was 75:25, and the flow rate was 0.5 mL min -1 The chromatographic column is a Waters Symmetry C18 chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, a filler particle size of 5 μm, a column temperature of 30°C, a detection wavelength of 322 nm, and a retention time of 9.8 min.

[0022] In step S3, the concentration of the NaOH solution is 1 mol L -1; The volume ratio of osthole extract and NaOH solution is 1:4.

[0023] Compared with the prior art, the present invention has the following beneficial effects.

[0024] 1. Traditional extraction methods usually use a large amount of toxic solvents to extract bioactive substances, and it is difficult to achieve purification and enrichment. In addition, in order to obtain the target substance, a time-consuming and energy-consuming solvent separation process is required. In contrast, the method proposed in the present invention is to extract the bioactive substance into a green natural low eutectic solvent, while enriching it into a slightly upgraded green convertible solvent, and the target substance can be quickly recovered by only simple pH adjustment.

[0025] 2. The present invention uses a natural low eutectic solvent synthesized from green renewable resources xylose, arabinose, glucose and levulinic acid as a green extraction solvent to replace traditional toxic solvents for the extraction of osthole, which greatly improves the extraction performance and reduces environmental pollution.

[0026] 3. The present invention uses a convertible solvent and a natural low eutectic solvent as an extractant to simultaneously complete extraction, purification and enrichment. Osthole is ultimately transferred to a microliter-level extractant, thereby achieving micro-amount and efficient extraction of osthole.

[0027] 4. The convertible solvent used in the present invention is pH responsive, so the conversion of its hydrophilicity and hydrophobicity can be achieved by simply adjusting the pH, and osthole is quickly separated and recovered, solving the problem of time-consuming and energy-consuming separation and recovery of traditional bioactive substances.

[0028] 5. The present invention not only realizes the recovery of osthole, but also recovers the natural low eutectic solvent while osthole is enriched in the convertible solvent. The recovered natural low eutectic solvent can be directly used to extract new osthole powder and can be recycled and reused multiple times, thus realizing the sustainable extraction of osthole. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 The effect of the type of natural deep eutectic solvent on the yield of osthole;

[0031] Figure 2 The effect of the molar ratio of each component in the natural deep eutectic solvent on the yield of osthole;

[0032] Figure 3 The effect of the water content of the natural deep eutectic solvent on the yield of osthole;

[0033] Figure 4 The effect of the ratio of Cnidium monnieri powder to natural deep eutectic solvent on the yield of Cnidium monnieri;

[0034] Figure 5 The effect of the type of convertible solvent on the yield of osthole;

[0035] Figure 6 is the effect of the volume of convertible solvent on the yield of osthole;

[0036] Figure 7 The effect of ultrasound temperature on the yield of osthole;

[0037] Figure 8 The effect of ultrasound time on the yield of osthole;

[0038] Fig. 9 The effect of the number of times the natural deep eutectic solvent is used on the recovery rate of osthole;

[0039] Fig.10 The effect of the number of times the natural deep eutectic solvent is used on the yield of osthole. DETAILED DESCRIPTION

[0040] Natural low eutectic solvents are a new type of green designable solvents, which are prepared from natural ingredients (sugars, alcohols, carboxylic acids and amino acids). They have the advantages of low toxicity, low volatility, biodegradability and easy preparation, and are expected to be used as green extractants to replace traditional toxic solvents. Liquid-liquid extraction can further purify and enrich bioactive substances, reduce impurity interference, and improve the purity and concentration of bioactive substances. Some convertible solvents can be reversibly converted between hydrophilicity and hydrophobicity by adjusting the pH value, and are expected to achieve rapid separation of target substances from the extractant. Therefore, the development of an extraction method combining a natural low eutectic solvent with a convertible solvent is of great significance for achieving simultaneous green extraction and enrichment of osthole and achieving rapid separation.

[0041] The present invention utilizes a natural low eutectic solvent and a convertible solvent to jointly extract and enrich osthole in Cnidium monnieri, and evaluates the recovery and repeated extraction capabilities of the natural low eutectic solvent. The present invention is innovatively designed to utilize a natural low eutectic solvent synthesized from renewable resources such as xylose, arabinose, glucose and levulinic acid to greenly extract osthole, while enriching the osthole in the natural low eutectic solvent extract into a slightly upgraded convertible solvent. By adjusting the pH to convert the hydrophobic convertible solution into a hydrophilic solution, osthole is quickly separated and recovered. The natural low eutectic solvent is recovered and directly used for new extraction, which solves the problems that the extractants used in the existing active substance extraction are highly toxic, large in dosage, and may cause environmental pollution, and that the recovery of the active substance requires a time-consuming and energy-consuming solvent separation process, thereby achieving sustainable extraction and separation of the active substance.

[0042] In order to better explain the present invention, so as to facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings. If not specifically indicated, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the reagents used can be commercially available products.

[0043] Example 1 Optimization of Extractant

[0044] A. Types of Natural Deep Eutectic Solvents

[0045] (1) Experimental method: 67 mg of Cnidium monnieri powder was accurately weighed into a 2 mL centrifuge tube, and 1 mL of a prepared natural low eutectic solvent (xylose-levulinic acid, arabinose-levulinic acid, glucose-levulinic acid) or methanol solution with a molar ratio of 1:4 and a water content of 50% was added, and 200 μL of a convertible solvent (heptanoic acid) was added. Ultrasonic extraction was performed at 30°C and a frequency of 40 Hz for 30 min. Osthole was extracted into the natural low eutectic solvent phase and then concentrated into the convertible solvent phase. The osthole extract was collected by centrifugation, and the yield of osthole was determined by Agilent 1260 HPLC-diode array chromatography. The detection conditions were: injection volume of 20 μL, mobile phase of methanol and water (75:25, v / v), flow rate of 0.5 mL min -1 The chromatographic column was a Waters Symmetry C18 column (150 mm × 4.6 mm, 5 μm), the column temperature was 30 ° C, the detection wavelength was 322 nm, and the retention time was 9.8 min.

[0046] (2) Experimental results: Figure 1 Different lowercase letters in the figure indicate significant differences at the 95% confidence level.

[0047] (3) Result analysis: The yields of osthole extracted by the three natural deep eutectic solvents were significantly higher than those by methanol, among which the natural deep eutectic solvent synthesized by xylose-levulinic acid had the highest yield of osthole. The reason may be that the natural deep eutectic solvents synthesized by different monosaccharides have different configurations and hydrogen bonding sites. Therefore, xylose-levulinic acid was selected as the best natural deep eutectic solvent.

[0048] B. Molar ratio of each component in natural deep eutectic solvent

[0049] (1) Experimental method: The same as method A in Example 1, except that different molar ratios (1:1, 1:2, 1:3, 1:4, 1:5, 1:6) were used to synthesize xylose-levulinic acid.

[0050] (2) Experimental results: Figure 2 Different lowercase letters in the figure indicate significant differences at the 95% confidence level.

[0051] (3) Result analysis: When the molar ratio is in the range of 1:1 to 1:4, the yield of osthole increases significantly. When the molar ratio exceeds 1:4, the yield of osthole decreases. This may be related to the fact that different molar ratios may affect the synthetic stability of natural deep eutectic solvents. Therefore, a molar ratio of 1:4 was selected to synthesize xylose-levulinic acid.

[0052] C. Water content of natural deep eutectic solvents

[0053] (1) Experimental method: The same as the method in Example 1A, except that natural deep eutectic solvents with different water contents (10, 30, 50, 60, 70%, v / v) were used.

[0054] (2) Experimental results: Figure 3 shown.

[0055] (3) Result analysis: When the water content is between 10% and 50%, the yield shows an upward trend. When the water content is 50%, the yield reaches the highest. When it exceeds 50%, the yield decreases significantly. The reason may be that the appropriate water content can significantly reduce the viscosity of the natural deep eutectic solvent and promote the diffusion of solutes into the solvent during the extraction process. Excessive water may destroy the hydrogen bonds between xylose and levulinic acid and reduce the interaction between the natural deep eutectic solvent and the target substance. Therefore, the optimal water content is selected as 50%.

[0056] D. Material-liquid ratio

[0057] (1) Experimental method: The same as the method A in Example 1, except that different material-liquid ratios (1:5, 1:8, 1:10, 1:15, 1:20 g mL -1 ) to extract osthole.

[0058] (2) Experimental results: Figure 4 shown.

[0059] (3) Result analysis: When the solid-liquid ratio was 1:5 to 1:15 g mL -1 The yield showed an upward trend within the range, and further increasing the volume of the natural deep eutectic solvent had no significant effect on the yield of osthole. Therefore, the material-liquid ratio was selected to be 1:15 g mL -1 .

[0060] E. Types of convertible solvents

[0061] (1) Experimental method: The same as the method in Example 1 A, except that different types of convertible solvents (hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid) were used to enrich osthole.

[0062] (2) Experimental results: Figure 5 shown.

[0063] (3) Result analysis: Among the four convertible solvents, heptanoic acid has the highest osthole yield. This may be related to the carbon chain length of the convertible solvent. The number of carbons on its alkyl group affects its hydrophilicity. Therefore, heptanoic acid was selected as the best convertible solvent.

[0064] F. Volume of convertible solvent

[0065] (1) Experimental method: The method was the same as that in Example 1A, except that different volumes of convertible solvent (100, 150, 200, 400, 600 μL) were used to enrich osthole.

[0066] (2) Experimental results: Figure 6 shown.

[0067] (3) Result analysis: When the volume of heptanoic acid was in the range of 100-200 μL, the yield increased. When the volume exceeded 200 μL, the yield did not change significantly. The target content in a small amount of extractant was easily saturated during the extraction process, and the target could not be fully enriched. However, as the volume of the extractant increased, the target could be completely extracted. Therefore, the optimal volume of heptanoic acid was selected to be 200 μL.

[0068] Example 2 Optimization of extraction conditions

[0069] A. Ultrasonic temperature

[0070] (1) Experimental method: The method is the same as that in Example 1A, except that different ultrasonic temperatures (20, 30, 40, 50, 60° C.) are used to extract osthole.

[0071] (2) Experimental results: Figure 7 shown.

[0072] (3) Result analysis: When the ultrasonic temperature is in the range of 20-30°C, the yield of osthole increases, and when it exceeds 30°C, the yield decreases significantly. Too high an ultrasonic temperature will lead to the degradation of bioactive substances, thereby reducing the yield of osthole. Therefore, the optimal ultrasonic temperature is selected to be 30°C.

[0073] B. Ultrasound time

[0074] (1) Experimental method: The method is the same as that in Example 1A, except that different ultrasonic times (10, 20, 30, 40, 50 min) were used to extract osthole.

[0075] (2) Experimental results: Figure 8 shown.

[0076] (3) Result analysis: When the ultrasonic time was within the range of 10 to 30 minutes, the yield of osthole increased significantly. However, the yield did not increase significantly when the ultrasonic time was further extended. Properly extending the extraction time was conducive to the full extraction of osthole. Therefore, the ultrasonic time of 30 minutes was selected.

[0077] Example 3 Recovery of Osthole

[0078] (1) Experimental method: 200 μL of the osthole heptanoic acid extract obtained in Example 1 was mixed with 800 μL of NaOH solution (1 mol L -1 ) were mixed in a 2 mL centrifuge tube. The solvent was converted from hydrophobic to hydrophilic, and the osthole solid adhered to the tube wall and was recovered. The recovery rate of osthole each time when the natural low eutectic solvent was reused multiple times was determined.

[0079] The recovered osthole solid was dissolved in 200 μL methanol. The content of the recovered osthole was then determined using the HPLC detection conditions for determining the yield of osthole. The recovery rate of osthole was calculated according to formula (1):

[0080] Recovery rate (%) = R / E × 100% (1)

[0081] Wherein R is the content of recovered osthole, and E is the content of extracted osthole.

[0082] (2) Experimental results: The results of using the same natural deep eutectic solvent for different times are as follows Fig. 9 shown.

[0083] (3) Result analysis: The natural deep eutectic solvent was reused 5 times, and the recovery rate of osthole remained above 90%. The slight decrease in the recovery rate may be due to the fact that the natural deep eutectic solvent contained more impurities after repeated use, and the convertible solvent could not fully purify them. Some impurities occupied the binding sites of osthole and the extractant, resulting in a decrease in the selectivity of the convertible solvent for the recovery of osthole.

[0084] Example 4 Recycling of natural deep eutectic solvent

[0085] (1) Experimental method: The natural deep eutectic solvent recovered in Example 1 was collected and directly used to extract new cnidium monnieri powder. The natural deep eutectic solvent was reused 5 times, and the yield of osthole extracted each time was measured.

[0086] (2) Experimental results: The results of using the same natural deep eutectic solvent for different times are as follows Fig.10 shown.

[0087] (3) Result analysis: After the osthole in the natural deep eutectic solvent in Example 1 is enriched into the convertible solution, the natural deep eutectic solvent can be directly used for new extraction. The results show that after the natural deep eutectic solvent is used 5 times, the yield of osthole does not change significantly. The synthesized natural deep eutectic solvent has good recyclability, which is conducive to the sustainable extraction of osthole.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A green and sustainable method for extracting and separating the biopesticide osthole, characterized in that: The steps include: S1, Preparation of natural deep eutectic solvent Xylose, arabinose and glucose are used as hydrogen bond donors, and levulinic acid is used as a hydrogen bond acceptor. The two components are mixed according to a preset molar ratio and heated until a uniform and transparent liquid is formed, thereby obtaining three natural low eutectic solvents of xylose-levulinic acid, arabinose-levulinic acid and glucose-levulinic acid. S2, extraction of osthole Accurately weigh the Cnidium monnieri powder into a centrifuge tube, add any one of xylose-levulinic acid, arabinose-levulinic acid, glucose-levulinic acid, and add a convertible solvent, perform ultrasonic simultaneous extraction and enrichment of osthole, extract the osthole into a natural low eutectic solvent phase, and concentrate it into a convertible solvent phase, then collect the convertible solvent phase, and determine the yield of osthole by high performance liquid chromatography; S3, recovery of osthole The osthole extract collected in step S2 is mixed with a NaOH solution to convert the solvent from hydrophobic to hydrophilic, and the osthole solid is precipitated and recovered; S4, reuse of natural deep eutectic solvents The natural deep eutectic solvent phase in step S2 is collected and directly used to extract new Cnidium monnieri powder.

2. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S1, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1 to 1:6; The water content of the natural deep eutectic solvent is 10 to 70%.

3. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S2, the material-liquid ratio of Cnidium monnieri powder to natural deep eutectic solvent is 1:5-1:20 g / mL -1 .

4. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S2, the solvent can be converted into any one of hexanoic acid, heptanoic acid, octanoic acid, and nonanoic acid.

5. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S2, the volume ratio of the natural deep eutectic solvent to the convertible solvent is 10:1 to 5:

3.

6. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S2, the ultrasonic temperature is 20-60°C, the ultrasonic frequency is 40 Hz, and the ultrasonic time is 10-50 min.

7. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S1, the hydrogen bond donor is xylose, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:4, and the water content of the natural deep eutectic solvent is 50%; In step S2, the material-liquid ratio of Cnidium monnieri powder to natural deep eutectic solvent is 1:15 g mL -1 , the convertible solvent is heptanoic acid, the volume ratio of the natural low eutectic solvent to the convertible solvent is 5:1, the ultrasonic temperature is 30°C, and the ultrasonic time is 30 min.

8. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1 or 7, characterized in that: In step S2, the yield of osthole was detected by Agilent 1260 high performance liquid chromatography-diode array, and the detection conditions were: injection volume of 20 μL, mobile phase of methanol and water, the volume ratio of methanol to water was 75:25, and the flow rate was 0.5 mL min -1 The chromatographic column is a Waters Symmetry C18 chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, a filler particle size of 5 μm, a column temperature of 30°C, a detection wavelength of 322 nm, and a retention time of 9.8 min.

9. The green and sustainable method for extracting and separating the biopesticide osthole according to claim 1, characterized in that: In step S3, the concentration of the NaOH solution is 1 mol L -1 ; The volume ratio of osthole extract and NaOH solution is 1:4.

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