Method for ultrasonic-assisted extraction of triterpenoid saponin in lonicera macranthoides by using deep-eutectic solvent

Abstract

The invention relates to a method for ultrasonically extracting triterpenoid saponin in lonicera macranthoides by using deep-eutectic solvents. The method specifically comprises the following steps: (1) preparing and screening 20 deep-eutectic solvents; (2) preparing lonicera macranthoides powder; (3) performing ultrasonic-assisted extraction on triterpenoid saponin in lonicera macranthoides by using the deep-eutectic solvent and the traditional solvent; (4) filtering the extracted lonicera macranthoides triterpenoid saponin solution, collecting filtrate, and detecting the content by HPLC (High Performance Liquid Chromatography); and (5) comparing with the traditional method. By adopting the method provided by the invention, the eutecticevaporate solvent with the best effect is used for extracting the macranthoidin B and the dipsacus asper saponin B in the macranthoidin, the total content can reach 101.82 mg / g, and the extraction efficiency is improved by 1.44-3.48 times compared with that of the traditional method. In general, the method has the advantages of being simple in preparation, good in safety, environmentally friendly, short in used time, high in extraction rate and the like, and the requirement of modern processing for environmental friendliness is met.

Description

Technical Field

[0001] The present invention relates to a method for ultrasonic extraction of macranthoidin B and asperosaponin VI from Lonicera macranthoides using a deep eutectic solvent, belonging to the technical field of active substance extraction. Background Art

[0002] Lonicera macranthoides is a semi-evergreen medicinal plant of the genus Lonicera in the family Caprifoliaceae, mainly distributed in Guizhou, Chongqing, Hunan and other provinces in southern China, and is an important local Chinese medicinal material resource. The dried and swollen flower buds and newly opened flowers of this plant are the main sources of the traditional Chinese medicine "Lonicera macranthoides", which is famous for its effects of clearing heat and detoxifying, and dispersing wind and heat.

[0003] The compounds extracted and separated from Lonicera macranthoides include triterpenoid saponins, phenylpropanoids, flavonoids, coumarin glycosides, monoterpene glycosides and saccharides, etc. Among them, the triterpenoid saponin components in Lonicera macranthoides are mainly macranthoidin B (MB) and asperosaponin VI (DB). Existing studies have shown that MB and DB have various biological activities such as anti-inflammatory, antioxidant, hepatoprotective, cholagogic and anti-tumor, making them have important application values in the fields of medicine, beverages, health products and daily chemicals.

[0004] In the field of extraction of active ingredients from traditional Chinese medicine, traditional methods often rely on organic solvents (such as methanol, ethanol, acetone) for extraction. However, these organic solvents have many disadvantages, such as being flammable, volatile, and easy to inhale and cause poisoning. Not only are there safety hazards during use, but large-scale use will also damage the natural environment and even threaten human health. Therefore, in order to achieve sustainable development and ensure human safety, it is of great significance to seek an "environmentally friendly solvent" that is easy to operate, has high efficiency and low energy consumption to replace traditional organic solvents for extraction. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art and provide a method for ultrasonic-assisted extraction of MB and DB from Lonicera macranthoides using a deep eutectic solvent and its application to improve the extraction efficiency of MB and DB. The purpose of the present invention can be achieved by the following technologies:

[0006] The first purpose of the present invention is to provide a method for extracting MB and DB from Lonicera macranthoides. Ultrasonic-assisted extraction of MB and DB from Lonicera macranthoides using a deep eutectic solvent is adopted, and the contents of MB and DB in different extraction solutions are determined by high performance liquid chromatography to determine the extraction solvent that maximizes the extraction rate of MB and DB from Lonicera macranthoides. The extraction method includes the following steps:

[0007] Step S1: Preparation of deep eutectic solvents: Mix the prepared hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:2, heat and stir until a colorless transparent solution is obtained, add water with a volume fraction of 40% after cooling, and a total of 20 deep eutectic solvents are obtained, and finally store them sealed;

[0008] Step S2: Preparation of Lonicera macranthoides Hand.-Mazz. powder: After drying Lonicera macranthoides Hand.-Mazz., crush it and pass through a 60-mesh sieve to obtain Lonicera macranthoides Hand.-Mazz. powder;

[0009] Step S3: Extraction of Lonicera macranthoides Hand.-Mazz. MB and DB: Mix the Lonicera macranthoides Hand.-Mazz. powder passing through a 60-mesh sieve with the prepared 20 deep eutectic solvents and traditional solvents (anhydrous ethanol and 50% ethanol) respectively, perform ultrasonic treatment, and the supernatant obtained after centrifugation and filtration is the extract of Lonicera macranthoides Hand.-Mazz. MB and DB, and perform HPLC analysis. Further, in Step S1, the conditions for heating and stirring are: stirring at a temperature of 70 - 80 °C for 1 - 2 h.

[0010] Further, in Step S1, the hydrogen bond acceptors include choline chloride, betaine, choline bromide, tetrapropylammonium bromide, and the hydrogen bond donors include urea, 1,4-butanediol, ethylene glycol, malic acid, citric acid, D,L-lactic acid, and the molar ratio of the acceptor to the donor is 1:2.

[0011] Further, in Step S3, the mass-volume ratio of Lonicera macranthoides Hand.-Mazz. powder to the deep eutectic solvent is 1 g:20 mL. Further, in Step S3, the temperature of ultrasonic extraction is 50 °C, the power is 280 W, and the time is 40 min. Further, in Step S3, the centrifugation conditions are a centrifugation speed of 4000 rpm and a centrifugation time of 15 min. The second object of the present invention is to provide an application of a deep eutectic solvent for extracting Lonicera macranthoides Hand.-Mazz. MB and DB, and the extraction efficiency obtained by using the method of extracting Lonicera macranthoides Hand.-Mazz. MB and DB with the deep eutectic solvent is 1.44 - 3.48 times higher than that of the traditional solvent extraction method.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] (1) The deep eutectic solvent of the present invention is simple to prepare, economical, and environmentally friendly. Using a one-step preparation method, two compounds are mixed until a homogeneous transparent liquid is formed, and the deep eutectic solvent is relatively stable to water and air.

[0014] (2) The ultrasonic extraction used in the present invention will not damage the structures of Lonicera macranthoides Hand.-Mazz. MB and DB, and the extraction is safe and has high extraction efficiency;

[0015] (3) The present invention uses high-performance liquid chromatography to screen out the optimal extraction solvent for extracting MB and DB from Lonicera macranthoides Hand.-Mazz. as choline chloride: ethylene glycol. And it is verified that the best deep eutectic solvent (choline chloride: ethylene glycol) has a higher extraction efficiency than traditional solvents and methods. Description of the Drawings

[0016] Figure 1 Standard curve of macranthoidin B (MB).

[0017] Figure 2 Standard curve of asperosaponin VI (DB).

[0018] Figure 3 Comparison of the extraction rates of MB and DB by 20 deep eutectic solvents and traditional solvents (anhydrous ethanol and 50% ethanol).

[0019] Figure 4 : HPLC chromatogram: (a) Traditional extraction method; (b) Ultrasonic-assisted deep eutectic solvent (choline chloride: ethylene glycol) extraction (DES-UAE); (c) Mixed standard. The peaks marked as 1 and 2 are MB and DB respectively. Figure 5 Comparison of different extraction methods (***, P < 0.001): Ultrasonic-assisted deep eutectic solvent (choline chloride: ethylene glycol) extraction (DES-UAE); Maceration extraction (ME); Percolation extraction (PE). Detailed Description of the Invention

[0020] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Unless otherwise specified, the equipment used in the following embodiments represents conventional equipment in the art; the reagents used, unless otherwise specified, represent commercially available products or are prepared by conventional methods in the art. Those not described in detail in the following embodiments can be achieved by conventional experimental means in the art.

[0021] In some typical embodiments of the present invention, the conditions for HPLC analysis include: using an Ascentis-C18 column with a particle size of 5 μm and an inner diameter of 250 mm × 4.6 mm.

[0022] In some typical embodiments of the present invention, the HPLC analysis conditions include: using acetonitrile as pump A and 0.4% acetic acid as pump B as the mobile phase;

[0023] The flow rate is 0.6 mL / min;

[0024] The injection volume is 20.0 μL;

[0025] Equipped with an evaporative light scattering detector.

[0026] In some typical embodiments of the present invention, the HPLC analysis conditions further include gradient elution:

[0027] 0 min: 10% A, 90% B;

[0028] 5 min: 20% A, 80% B;

[0029] 10 min: 40% B, 60% A;

[0030] 30 min: 65% B, 35% A;

[0031] 31 min: 10% A, 90% B

[0032] 45 min: 10% A, 90% B.

[0033] Examples

[0034] Preparation of deep eutectic solvents:

[0035] All chemical reagents, including choline chloride, betaine, choline bromide, tetrapropylammonium bromide, urea, 1,4-butanediol, ethylene glycol, malic acid, citric acid, D,L-lactic acid, were weighed according to a fixed molar ratio of hydrogen bond acceptor: hydrogen bond donor = 1:2 (as shown in Table 1), and then mixed in a beaker. The beaker was placed in a water bath on a magnetic stirrer and heated until a homogeneous and transparent liquid was obtained. After standing and cooling, water with a volume fraction of 40% was added.

[0036] Table 1 Twenty kinds of DESs prepared

[0037]

[0038]

[0039] The deep eutectic solvents prepared above were placed in a conical flask for standby.

[0040] Establishment of standard curves for macranthoidin B (MB) and asperosaponin VI (DB):

[0041] The MB standard (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.) was diluted with anhydrous methanol solution to prepare control solutions with concentrations of 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L. The DB (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.) was diluted with anhydrous methanol solution to prepare control solutions with concentrations of 0.02 mg / L, 0.04 mg / L, 0.06 mg / L, 0.08 mg / L, and 0.10 mg / L. The peak areas were determined by HPLC. Using the MB concentration as the abscissa and the peak area as the ordinate, the standard curve of the MB reference substance was plotted ( Figure 1 as shown), and the standard curve was Y MB = 4174.3X – 471.67 (R2 = 0.9992); using the DB concentration as the abscissa and the peak area as the ordinate, the standard curve of the DB reference substance was plotted ( Figure 2 as shown), and the standard curve was Y DB = 4340.5X – 36.01 (R2 = 0.9993).

[0042] This example was to establish the standard curves of MB and DB, providing a judgment standard for the content determination of the main saponins of Lonicera macranthoides in the extract, so as to screen out the best eutectic solvent for extracting MB and DB from Lonicera macranthoides. Preparation of 20 eutectic solvent extracts of Lonicera macranthoides and extracts of Lonicera macranthoides with traditional solvents (anhydrous ethanol and 50% ethanol):

[0043] Accurately weigh 1.0 g of sieved Lonicera macranthoides powder. According to the mass-volume ratio of Lonicera macranthoides powder to the eutectic solvent of 1 g∶20 mL, the water content of the eutectic solvent of 40% (V / V), and the molar ratio of hydrogen bond acceptor to hydrogen bond donor of 1:2, the Lonicera macranthoides powder was respectively mixed evenly with the prepared 20 eutectic solvents and traditional solvents (anhydrous ethanol and 50% ethanol). After ultrasonic-assisted extraction, the extraction temperature was 50 °C, the extraction time was 40 min, and the ultrasonic power was 280 W; after the reaction, centrifugation was carried out, and the supernatant was taken to obtain 20 eutectic solvent extracts of Lonicera macranthoides and extracts of Lonicera macranthoides with traditional solvents (anhydrous ethanol and 50% ethanol) respectively.

[0044] Screening of eutectic solvents:

[0045] The obtained 20 eutectic solvent extracts of Lonicera macranthoides and extracts of Lonicera macranthoides with traditional solvents (anhydrous ethanol and 50% ethanol) were filtered through a 0.45 μm organic membrane to obtain sample solutions, and HPLC detection was carried out. The chromatograms are as Figure 4 shown. According to the obtained standard curve, the mass of the main saponins of Lonicera macranthoides in the extract was calculated, and further, the extraction rates of MB and DB in Lonicera macranthoides were calculated according to formula (1). The extraction rate calculation formulas of MB and DB are as follows:

[0046]

[0047] Chromatographic conditions:

[0048] The extract solutions of Lonicera macranthoides samples obtained with deep eutectic solvents and traditional solvents (absolute ethanol and 50% ethanol) were subjected to HPLC analysis. The analysis conditions were as follows: using an Ascentis-C18 column with a particle size of 5 μm and an inner diameter of 250 mm × 4.6 mm; using acetonitrile as pump A and 0.4% acetic acid as pump B as the mobile phase, with a flow rate of 0.6 mL / min, equipped with an evaporative light scattering detector, and an injection volume of 20.0 μL. The detailed elution steps are shown in Table 2.

[0049] Table 2 HPLC elution gradient

[0050]

[0051]

[0052] As Figure 3 shown, the effects of 20 deep eutectic solvents (DESs) and traditional solvents (absolute ethanol and 50% ethanol) on the extraction rates of Lonicera macranthoides MB and DB were evaluated. The results showed that among all the deep eutectic solvents, choline chloride: ethylene glycol (1:2, 40% water content) had the best effect, and the extraction rates of MB and DB reached 101.82 mg / g, which was better than other deep eutectic solvents. Among the traditional solvents, 50% ethanol obtained the highest extraction rate, and the extraction rates of MB and DB were 70.83 mg / g. However, this extraction rate was still lower than that of the best deep eutectic solvent. This difference may be related to the higher solubility of MB and DB in deep eutectic solvents and the hydrogen bond interaction between deep eutectic solvents and saponins, which improved the extraction efficiency.

[0053] Maceration extraction (ME):

[0054] Accurately weigh 1.0 g of sieved Lonicera macranthoides powder. According to the mass-volume ratio of Lonicera macranthoides powder to 50% ethanol of 1 g∶20 mL, at 25 °C, macerate the sample for 6 hours, and then centrifuge at a speed of 4000 r / min for 15 minutes. Discard the precipitate, and the obtained extract is filtered through a 0.45 μm microporous membrane for HPLC detection. Percolation extraction (PE):

[0055] Accurately weigh 4.0 g of sieved Lonicera macranthoides powder and put it into a glass percolator. Soak it with 50% ethanol for 12 hours, and perform percolation at a flow rate of 3 mL / min, with the temperature maintained at 25 °C. Collect the filtrate, filter it through a 0.45 μm microporous membrane for HPLC detection.

[0056] The key to the ultrasonic extraction process is the cavitation effect. Ultrasonic waves cause the formation and eventual rupture of bubbles in the liquid by generating alternating compression and rarefaction in the medium molecules, thereby destroying the cell structure and promoting the dissolution of target compounds. As Figure 5 shown, the extraction efficiency of ultrasonic-assisted deep eutectic solvent extraction is 3.45 times higher than that of maceration extraction and 2.46 times higher than that of percolation extraction. Another reason why ultrasonic-assisted deep eutectic solvent extraction has a higher extraction rate than traditional methods is that cellulose is the main component of plant cell walls. Compared with traditional solvents, deep eutectic solvents can effectively decompose cellulose, thus achieving efficient cellulose dissolution. The HBA and HBD in the deep eutectic solvent interact with the cellulose chains, significantly enhancing the dissolution process. This synergistic effect breaks the hydrogen bonds in the cellulose structure, loosens its dense network, and ultimately leads to complete dissolution.

[0057] In the present invention, the optimal deep eutectic solvent is screened by using high-performance liquid chromatography to determine the contents of MB and DB in the extracts of 20 deep eutectic solvents and traditional solvents, and then the deep eutectic solvent with the best effect is screened out.

[0058] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of low eutectic solvents, characterized in that: It includes the following steps: Step S1: Preparation of low eutectic solvents: Mix the prepared hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:2, heat and stir until a colorless and transparent solution is obtained, add 40% by volume of water after cooling, and obtain 20 kinds of low eutectic solvents in total, and finally seal and store; Step S2.: Preparation of gray felt honeysuckle powder: drying the gray felt honeysuckle, and then crushing it through a 60-mesh sieve to obtain gray felt honeysuckle powder; Step S3: Extraction of triterpenoid saponins from Lonicera japonica: The Lonicera japonica powder that had passed through a 60-mesh sieve was mixed with 20 prepared low eutectic solvents respectively, subjected to ultrasonic treatment, and centrifuged and filtered to obtain a supernatant, which was the triterpenoid saponin extract from Lonicera japonica, for HPLC analysis.

2. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of the deep eutectic solvent according to claim 1, characterized in that: The heating and stirring conditions in step S1 are: stirring at a temperature of 70-80° C. for 1-2 h.

3. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of the deep eutectic solvent according to claim 1, characterized in that: In step S1, the hydrogen bond acceptors include choline chloride, betaine, choline bromide, and tetrapropylammonium bromide; the hydrogen bond donors include urea, 1,4-butanediol, ethylene glycol, malic acid, citric acid, and D,L-lactic acid.

4. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 1, characterized in that: In step S3, the mass volume ratio of the gray felt honeysuckle powder and the low eutectic solvent is 1g:20mL.

5. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 1, characterized in that: The ultrasonic treatment conditions in step S3 are as follows: ultrasonic time 40 min, ultrasonic temperature 50° C., and ultrasonic power 280 W.

6. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 1, characterized in that: The centrifugal conditions in step S3 are a centrifugal speed of 4000 rpm and a centrifugal time of 15 min.

7. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 1, characterized in that: The conditions of the HPLC analysis include: using an Ascentis-C18 column with a particle size of 5 μm and an inner diameter of 250 mm×4.6 mm.

8. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 7, characterized in that: The HPLC analysis conditions include: using acetonitrile as A pump and 0.4% acetic acid B pump as mobile phase; The flow rate was 0.6 mL / min; The injection volume was 20.0 μL; Equipped with an evaporative light scattering detector.

9. The method for extracting triterpenoid saponins from Lonicera japonica by ultrasound-assisted extraction of deep eutectic solvent according to claim 8, characterized in that: The HPLC analysis conditions also include gradient elution: 0min: 10% A, 90% B; 5min: 20% A, 80% B; 10 min: 40% B, 60% A; 30min: 65% B, 35% A; 31min: 10% A, 90% B 45min: 10% A, 90% B.

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