Preparation method and application of a seven-membered cucurbituril supermolecular co-crystal solvent

Abstract

This invention discloses a method for preparing a seven-membered cucurbita supramolecular cocrystal solvent and its application. The invention uses a seven-membered cucurbita as a hydrogen bond acceptor and malic acid as a hydrogen bond donor to prepare a seven-membered cucurbita supramolecular cocrystal solvent. This solvent can be used to extract camptothecin from Camptotheca acuminata fruit, and the extracted camptothecin can be determined by HPLC. This seven-membered cucurbita supramolecular cocrystal solvent is a green extraction solvent that does not use organic solvents when extracting active ingredients from plants, while the extracted active ingredients retain their intrinsic activity. The preparation method is simple, the extraction efficiency is high, and efficient, green extraction is achieved.

Description

Technical Field

[0001] This invention relates to the field of eutectic solvent technology, and in particular to a method for preparing a seven-membered cucurbitacinoid supramolecular eutectic solvent and its application. Background Technology

[0002] SupraDES (supramolecular cocrystal solvent) is a green solvent system that combines supramolecular inclusion and deep cocrystal properties. Its formation depends on the synergistic effect of hydrogen bond networks and supramolecular host-guest interactions. This solvent system exhibits high efficiency, mildness and green technical advantages in the extraction of natural active ingredients. Through selective interaction with target components, it significantly improves extraction efficiency and product purity, while avoiding the toxicity and environmental pollution problems of traditional organic solvents.

[0003] The seven-membered cucurbit (Q[7]) is a supramolecular macrocyclic host compound with a highly symmetrical cage-like cavity structure and dense carbonyl oxygen ports. It can specifically recognize and encapsulate guest molecules of suitable size, exhibiting excellent selectivity and stability in the field of adsorption separation. Moreover, it has good biocompatibility, which is in line with the concept of green chemistry development.

[0004] Currently, there are no reports on seven-membered cucurbitacinol supramolecular cocrystal solvents. Therefore, this invention prepares a seven-membered cucurbitacinol supramolecular cocrystal solvent using the seven-membered cucurbitacinol as a hydrogen bond acceptor and malic acid as a hydrogen bond donor. By utilizing the molecular recognition ability of the seven-membered cucurbitacinol in the system for the target compound, efficient separation of the target component from matrix impurities is achieved. Simultaneously, the excellent solubility and mild reaction environment of the cocrystal solvent ultimately achieve the technical goals of improving extraction rate, increasing product purity, and reducing environmental risks. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a seven-membered cucurbita supramolecular cocrystal solvent and its application. This invention uses a seven-membered cucurbita as a hydrogen bond acceptor and malic acid as a hydrogen bond donor to prepare a seven-membered cucurbita supramolecular cocrystal solvent. This solvent is a green extraction solvent that does not use organic solvents when extracting active ingredients from plants, while the extracted active ingredients retain their intrinsic activity. The preparation method is simple, has high extraction efficiency, and achieves efficient and green extraction.

[0006] To solve the above technical problems, the present invention adopts the following technical solution: A method for preparing a seven-membered cucurbitacin supramolecular cocrystal solvent involves using malic acid as a hydrogen bond donor and a seven-membered cucurbitacin as a hydrogen bond acceptor. Malic acid and the seven-membered cucurbitacin are weighed and mixed at a mass ratio of 8-12:1. Then, water is added to the mixture, which is 25-35% of the total mass of the malic acid and the seven-membered cucurbitacin. The mixture is heated and stirred at 80-100℃ for 30-50 minutes to obtain a homogeneous and transparent solution, which is the seven-membered cucurbitacin supramolecular cocrystal solvent.

[0007] The aforementioned method for preparing the seven-membered cucurbitacin supramolecular cocrystal solvent involves using malic acid as a hydrogen bond donor and a seven-membered cucurbitacin as a hydrogen bond acceptor. Malic acid and the seven-membered cucurbitacin are weighed and mixed in a mass ratio of 9-11:1. Then, water is added to the mixture, which is 25-30% of the total mass of the malic acid and the seven-membered cucurbitacin. The mixture is heated and stirred at 85-95°C for 35-45 minutes to obtain a uniform and transparent solution, which is the seven-membered cucurbitacin supramolecular cocrystal solvent.

[0008] Specifically, the aforementioned method for preparing the seven-membered cucurbita supramolecular cocrystal solvent involves using malic acid as a hydrogen bond donor and a seven-membered cucurbita as a hydrogen bond acceptor. Malic acid and the seven-membered cucurbita are weighed and mixed in a mass ratio of 10:1. Then, water equal to 25% of the total mass of malic acid and the seven-membered cucurbita is added. After heating and stirring at 90°C for 40 minutes, a uniform and transparent solution is obtained, which is the seven-membered cucurbita supramolecular cocrystal solvent.

[0009] The aforementioned application of the seven-membered cucurbita supramolecular eutectic solvent in the determination of camptothecin content in Camptotheca acuminata fruit involves using the seven-membered cucurbita supramolecular eutectic solvent as the extraction solvent to extract camptothecin from Camptotheca acuminata fruit, and then using HPLC to determine the content of the extracted camptothecin.

[0010] For the aforementioned applications, the content determination shall be performed according to the following steps: (1) Pretreatment of Camptotheca acuminata fruit: Place Camptotheca acuminata fruit in an oven at 45-55℃ and dry for 7-9 hours. After drying, put it into a pulverizer and pulverize it. Then pass it through a 30-60 mesh sieve to obtain Camptotheca acuminata fruit powder for later use. (2) Extraction of Camptotheca acuminata fruit: Accurately weigh 40-50 mg of Camptotheca acuminata fruit powder, add 250-450 μL of seven-membered cucurbita ring supramolecular eutectic solvent and 750-550 μL of purified water, and extract by ultrasonication at 30-45℃ for 5-30 min. Then add purified water to the total volume of 3 mL, shake evenly and centrifuge for 8-12 min. Take the supernatant and filter to obtain Camptotheca acuminata fruit extract. (3) Preparation of standard solutions: Weigh the camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a standard stock solution with a concentration of 1 mg / mL; then dilute stepwise with methanol to prepare standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL, respectively, for later use; (4) Preparation of standard curve: Standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL were analyzed by HPLC at concentrations from low to high. The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The chromatographic conditions were as follows: Chromatographic column: C18 Superb 5um, 4×150mm; mobile phase: methanol-water, methanol to water volume ratio: 45:55, flow rate: 1mL / min, injection volume: 20μL; detection wavelength: 254nm; column temperature: 30℃. (5) Content determination: Take the extract of Camptotheca acuminata fruit and perform HPLC analysis and detection. The chromatographic conditions are the same as in step (4). The obtained peak area is substituted into the standard curve to calculate the corresponding concentration.

[0011] In the aforementioned step (1), the pretreatment of Camptotheca acuminata fruit is as follows: the Camptotheca acuminata fruit is placed in an oven at 48-52℃ and dried for 7.5-8.5 hours. After drying, it is placed in a pulverizer and pulverized. Then it is passed through a 40-50 mesh sieve to obtain Camptotheca acuminata fruit powder for later use.

[0012] Specifically, in the aforementioned step (1), the pretreatment of Camptotheca acuminata fruit is as follows: the Camptotheca acuminata fruit is placed in an oven at 50°C and dried for 8 hours. After drying, it is placed in a pulverizer and pulverized. Then it is passed through a 40-mesh sieve to obtain Camptotheca acuminata fruit powder for later use.

[0013] In step (2) above, extracting Camptotheca acuminata fruit: accurately weigh 50-65 mg of Camptotheca acuminata fruit powder, add 250-350 μL of seven-membered cucurbita supramolecular eutectic solvent and 750-650 μL of purified water, and extract by ultrasonication at 40-45℃ for 5-15 min. Then add purified water to a total volume of 3 mL, shake evenly, centrifuge for 9-11 min, take the supernatant and filter to obtain Camptotheca acuminata fruit extract.

[0014] Specifically, in step (2) above, the camptotheca fruit is extracted: accurately weigh 50 mg of camptotheca fruit powder, add 250 μL of seven-membered cucurbita supramolecular eutectic solvent and 750 μL of purified water, and extract by ultrasonication at 40°C for 15 min. Then add purified water to a total volume of 3 mL, shake evenly and centrifuge for 10 min, take the supernatant and filter to obtain the camptotheca fruit extract.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The seven-membered cucurbita ring supramolecular eutectic solvent prepared by this invention is a green extraction solvent that does not use organic solvents when extracting active ingredients from plants and will not have an impact on the environment.

[0016] Using a seven-membered cucurbitacin supramolecular eutectic solvent as the extraction solvent results in a short extraction time and a low extraction temperature. This prevents the extraction of active ingredients from being destroyed due to excessive time or temperature, and the extracted active ingredients retain their intrinsic activity. When using a seven-membered cucurbitacin supramolecular eutectic solvent to extract Camptotheca acuminata fruit, the extraction time is only 10 minutes, and the extraction temperature is between 40-45℃, which is sufficient for complete extraction. The anticancer activity of the Camptotheca acuminata fruit extract is not affected.

[0017] The preparation method of the seven-membered cucurbitacin supramolecular eutectic solvent is simple and has high extraction efficiency. It can utilize the molecular recognition ability of the seven-membered cucurbitacin to achieve efficient and green extraction of target compounds. Compared with traditional solvents, it has significant advantages such as high extraction rate, environmental friendliness and simple operation. Attached Figure Description

[0018] Figure 1 A schematic diagram of the solvent synthesis of a seven-membered cucurbitacin supramolecular eutectic; Figure 2 For Q[7], MA and Q[7] / MA 1 H NMR spectrum; Figure 3 Infrared spectra of Q[7], MA, and Q[7] / MA; Figure 4 XRD patterns of Q[7], MA, and Q[7] / MA; Figure 5 The viscosity plot is Q[7] / MA; Figure 6 Density plot of Q[7] / MA; Figure 7 The effects of different Q[7] / MA contents, material-liquid ratios, ultrasonic time, and extraction temperatures on the extraction efficiency of camptothecin were investigated. Figure 8 A comparison chart of the extraction rates of Q[7] / MA, CA / MA and 75% methanol (100:1 HCl); Figure 9 Cell viability (%, mean ± SD, n = 6) of 4T1 cells treated with different concentrations of CPT; Figure 10 The dose-response curve of CPT on 4T1 cells is shown. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0020] Unless otherwise specified, the chemical reagents used in the embodiments of this invention are all commercially available products commonly used in the art. Equivalent substitutes for the reagents used in this invention are considered to fall within the protection scope of this invention as long as they meet the technical specifications.

[0021] Example 1 Preparation method of seven-membered cucurbitacin supramolecular eutectic solvent: Malic acid (MA) was used as a hydrogen bond donor and cucurbitacin (Q[7]) was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed in a mass ratio of 10:1, mixed, and then water was added in a mass ratio of 25% of the total mass of malic acid and cucurbitacin. After heating and stirring at 90°C for 40 min, a uniform and transparent solution was obtained, which is the cucurbitacin supramolecular eutectic solvent (Q[7] / MA).

[0022] Example 2 Preparation method of seven-membered cucurbitacin supramolecular eutectic solvent: Malic acid was used as a hydrogen bond donor and cucurbitacin was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed in a mass ratio of 8:1, mixed, and then water was added in a mixture equal to 35% of the total mass of malic acid and cucurbitacin. The mixture was heated and stirred at 100°C for 30 minutes to obtain a uniform and transparent solution, which is the supramolecular eutectic solvent of cucurbitacin.

[0023] Example 3 Preparation method of seven-membered cucurbitacin supramolecular eutectic solvent: Malic acid was used as a hydrogen bond donor and cucurbitacin was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed in a mass ratio of 12:1, mixed, and then water was added in a mixture equal to 25% of the total mass of malic acid and cucurbitacin. The mixture was heated and stirred at 80°C for 50 minutes to obtain a homogeneous and transparent solution, which is the supramolecular eutectic solvent of cucurbitacin.

[0024] Example 4 Preparation method of seven-membered cucurbitacin supramolecular eutectic solvent: Malic acid was used as a hydrogen bond donor and cucurbitacin was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed at a mass ratio of 9:1, mixed, and then water was added at a mass ratio of 33% of the total mass of malic acid and cucurbitacin. After heating and stirring at 85°C for 35 minutes, a uniform and transparent solution was obtained, which is the supramolecular eutectic solvent of cucurbitacin.

[0025] Example 5 Preparation method of seven-membered cucurbitacin supramolecular eutectic solvent: Malic acid was used as a hydrogen bond donor and cucurbitacin was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed in a mass ratio of 11:1, mixed, and then water was added in a mixture equal to 27% of the total mass of malic acid and cucurbitacin. The mixture was heated and stirred at 95°C for 45 minutes to obtain a homogeneous and transparent solution, which is the supramolecular eutectic solvent of cucurbitacin.

[0026] Example 6 A method for measuring the camptothecin content in Camptotheca acuminata fruit using a seven-membered cucurbitacin supramolecular eutectic solvent as the extraction solvent: (1) Pretreatment of Camptotheca acuminata fruit: Place Camptotheca acuminata fruit in an oven at 50°C for 8 hours. After drying, place it in a pulverizer to pulverize it and then pass it through a 40-mesh sieve to obtain Camptotheca acuminata fruit powder for later use. (2) Extraction of Camptotheca acuminata fruit: Accurately weigh 50 mg of Camptotheca acuminata fruit powder, add 250 μL of seven-membered cucurbita supramolecular eutectic solvent and 750 μL of purified water, and extract by ultrasonication at 40℃ for 15 min. Then add purified water to the total volume of 3 mL, shake evenly and centrifuge for 10 min. Take the supernatant and filter to obtain Camptotheca acuminata fruit extract. (3) Preparation of standard solutions: Weigh the camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a standard stock solution with a concentration of 1 mg / mL; then dilute stepwise with methanol to prepare standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL, respectively, for later use; (4) Preparation of standard curve: Standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL were analyzed by HPLC at concentrations from low to high. The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The chromatographic conditions were as follows: Chromatographic column: C18 Superb 5um, 4×150mm; mobile phase: methanol-water, methanol to water volume ratio: 45:55, flow rate: 1mL / min, injection volume: 20 μL; detection wavelength: 254nm; column temperature: 30℃. (5) Content determination: Take the extract of Camptotheca acuminata fruit and perform HPLC analysis and detection. The chromatographic conditions are the same as in step (4). The obtained peak area is substituted into the standard curve to calculate the corresponding concentration.

[0027] Example 7 A method for measuring the camptothecin content in Camptotheca acuminata fruit using a seven-membered cucurbitacin supramolecular eutectic solvent as the extraction solvent: (1) Pretreatment of Camptotheca acuminata fruit: Place Camptotheca acuminata fruit in an oven at 55℃ for 7 hours. After drying, place it in a pulverizer to pulverize it and then pass it through a 60-mesh sieve to obtain Camptotheca acuminata fruit powder for later use. (2) Extraction of Camptotheca acuminata fruit: Accurately weigh 50 mg of Camptotheca acuminata fruit powder, add 450 μL of seven-membered cucurbita supramolecular eutectic solvent and 750 μL of purified water, and extract by ultrasonication at 45℃ for 5 min. Then add purified water to the total volume of 3 mL, shake evenly and centrifuge for 12 min. Take the supernatant and filter to obtain Camptotheca acuminata fruit extract. (3) Preparation of standard solutions: Weigh the camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a standard stock solution with a concentration of 1 mg / mL; then dilute stepwise with methanol to prepare standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL, respectively, for later use; (4) Preparation of standard curve: Standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL were analyzed by HPLC at concentrations from low to high. The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The chromatographic conditions were as follows: Chromatographic column: C18 Superb 5um, 4×150mm; mobile phase: methanol-water, methanol to water volume ratio: 45:55, flow rate: 1mL / min, injection volume: 20 μL; detection wavelength: 254nm; column temperature: 30℃. (5) Content determination: Take the extract of Camptotheca acuminata fruit and perform HPLC analysis and detection. The chromatographic conditions are the same as in step (4). The obtained peak area is substituted into the standard curve to calculate the corresponding concentration.

[0028] Example 8 A method for measuring the camptothecin content in Camptotheca acuminata fruit using a seven-membered cucurbitacin supramolecular eutectic solvent as the extraction solvent: (1) Pretreatment of Camptotheca acuminata fruit: Place Camptotheca acuminata fruit in an oven at 45℃ and dry for 9 hours. After drying, put it into a pulverizer and pulverize it. Then pass it through a 30-mesh sieve to obtain Camptotheca acuminata fruit powder for later use. (2) Extraction of Camptotheca acuminata fruit: Accurately weigh 40 mg of Camptotheca acuminata fruit powder, add 250 μL of seven-membered cucurbita supramolecular eutectic solvent and 550 μL of purified water, and extract by ultrasonication at 30℃ for 30 min. Then add purified water to the total volume of 3 mL, shake evenly and centrifuge for 8 min. Take the supernatant and filter to obtain Camptotheca acuminata fruit extract. (3) Preparation of standard solutions: Weigh the camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a standard stock solution with a concentration of 1 mg / mL; then dilute stepwise with methanol to prepare standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL, respectively, for later use; (4) Preparation of standard curve: Standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL were analyzed by HPLC at concentrations from low to high. The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The chromatographic conditions were as follows: Chromatographic column: C18 Superb 5um, 4×150mm; mobile phase: methanol-water, methanol to water volume ratio: 45:55, flow rate: 1mL / min, injection volume: 20 μL; detection wavelength: 254nm; column temperature: 30℃. (5) Content determination: Take the extract of Camptotheca acuminata fruit and perform HPLC analysis and detection. The chromatographic conditions are the same as in step (4). The obtained peak area is substituted into the standard curve to calculate the corresponding concentration.

[0029] To obtain the solution of this invention and verify its technical effects, the inventors conducted extensive experimental research, some of which are recorded below: 1. Preparation of supramolecular eutectic solvents Malic acid (MA) was weighed as a hydrogen bond donor and cucurbitacin (Q[7]) as a hydrogen bond acceptor in a mass ratio of 10:1. Then, 25% (w / w) of water was added to the mixture. After heating and stirring at 90°C for 40 min, a uniform and transparent solution was obtained, which is the supramolecular eutectic solvent (Q[7] / MA). The solution was then allowed to cool naturally to room temperature and stored for later use.

[0030] 2. Characterization of supramolecular eutectic solvents 2.1 Supramolecular eutectic solvent 1 H NMR spectrum Figure 2 In 1The H NMR spectrum shows that the characteristic peaks of the seven-membered cucurbita ring (Q[7]) are mainly distributed in the range of 4.0-6.0 ppm, which are attributed to the hydrogen signals of the methylene group at the port and the methylene group on the ring; while the characteristic peaks of malic acid (MA) are concentrated in two ranges: 2.50-3.00 ppm and 4.0-5.00 ppm, corresponding to the methylene hydrogen and methine hydrogen signals, respectively. Among them, the multiple peaks at 2.50-3.00 ppm are from the -CH2- hydrogen adjacent to the carboxyl group in MA, which splits due to the spin coupling between it and the adjacent methine hydrogen; the peaks in the range of 4.0-5.00 ppm correspond to the -CH(OH)-methine hydrogen of MA, which appears in the mid-chemical shift region due to the electronic effect of the hydroxyl and carboxyl groups. The green spectral line is the H NMR signal of the cocrystal solvent formed by Q[7] and MA. Its overall peak position retains the main characteristics of Q[7] and MA, but some peaks are broadened, the chemical shifts are slightly shifted, and the peak intensity distribution is different from that of the pure product. These changes reflect the influence of intermolecular interactions on the hydrogen chemical environment in the eutectic solvent. Further comparison of the spectra of pure Q[7], pure MA, and Q[7] / MA eutectic solvent reveals that the characteristic peak of MA shifts slightly to a higher field in the eutectic solvent, while the port hydrogen peak of Q[7] also shows a slight shift, which is mainly attributed to the formation of hydrogen bonds within the system. The above NMR spectral characteristics fully confirm that Q[7] and MA have successfully formed a deep eutectic solvent.

[0031] 2.2 Infrared spectrum of supramolecular eutectic solvent Figure 3 In the infrared spectrum, the absorption peaks of the eutectic solvent generally inherit the core functional group characteristics of Q[7] and MA, but the key peak positions are shifted and the peak shapes are also changed, which is a direct manifestation of the intermolecular interaction after the two form a eutectic solvent. In MA, the hydroxyl stretching vibration is at 3479 cm⁻¹ -1 A stretching vibration peak of -OH appears at 1734 cm⁻¹. Due to the hydrogen bonding between hydroxyl groups, the peak is broad and strong. -1 The peak at 1103 cm⁻¹ represents the stretching vibration of the C=O group of the carboxyl group. -1 The peak of the stretching vibration of -CO- is at 1741 cm⁻¹; Q[7] is at 1741 cm⁻¹. -1 The peak at 1475 cm⁻¹ is the characteristic absorption of the carbonyl group at its port. -1 It is the bending vibration of methylene-CH2- on the cucurbit ring cavity framework. The hydroxyl stretching vibration peak in the eutectic solvent (Q[7] / MA) shifts to 3477 cm⁻¹. -1 The peak shifted to a lower wavenumber and broadened, indicating that the hydroxyl group of MA formed an intermolecular hydrogen bond with the carbonyl group at the Q[7] port -C=O, resulting in a decrease in the OH bond energy and vibrational frequency. The carbonyl peak shifted to 1732 cm⁻¹. -1 A slight redshift and decreased peak intensity indicate CO single bond stretching vibration; the peak shifts to 1101 cm⁻¹.-1 Slight redshift, while Q[7] at 1475 cm -1 The characteristic peak shifts to 1479 cm⁻¹ in the complex. -1 This also corroborates that Q[7] forms a hydrogen bond with MA. The low wavenumber redshift, peak broadening and peak intensity change of multiple characteristic peaks in the spectrum clearly prove that a hydrogen bond is formed between Q[7] and MA, indicating the success of the deep eutectic solvent preparation.

[0032] 2.3 X-ray electron spectra (XRD) of eutectic solvents Figure 4 In the XRD pattern, MA exhibits sharp and dense diffraction peaks, mainly distributed in the 10-20°2θ range. This is a typical XRD characteristic of crystalline MA, reflecting that its molecules are arranged in a highly ordered crystalline form in the solid state, with strong long-range order. The diffraction peaks of Q[7] are broadened diffuse scattering peaks, with peak intensities much lower than MA, and no sharp characteristic crystalline peaks. This indicates that Q[7] is an amorphous / low-crystallinity structure under the test conditions, with poor long-range order of molecular arrangement and only short-range local order. The diffraction curve of the eutectic solvent is a broadened bulging background peak, completely eliminating the sharp crystalline peaks of MA. The peak shape is similar to the amorphous peak shape of Q[7] but flatter, indicating that after the formation of the deep eutectic solvent, the crystalline structure of MA is destroyed, and it changes from a crystalline state to an amorphous state. The XRD pattern confirms that Q[7] and MA formed a deep eutectic solvent.

[0033] 3. Physicochemical properties of eutectic solvents The viscosity and density of the prepared eutectic solvent were investigated. Figure 5 and Figure 6 It can be seen that the viscosity and density of the eutectic solvent both decrease significantly with increasing temperature, which means that increasing the temperature can increase the mass transfer rate of the eutectic solvent. The content of camptothecin in camptothecin fruit was determined by HPLC coupled with the prepared eutectic solvent. 4.1 Extraction Steps First, dry the camptotheca fruit in an oven at 50°C for 8 hours. After drying, grind it in a grinder, then pass it through a 40-mesh sieve and set it aside for later use.

[0034] Add 250 μL of seven-membered cucurbita supramolecular cocrystal solvent to 750 μL of purified water to obtain a 25% seven-membered cucurbita supramolecular cocrystal solvent. Then accurately weigh 50 mg of Camptotheca acuminata fruit and add it to the 25% seven-membered cucurbita supramolecular cocrystal solvent. Extract by sonication at 42℃ for 15 min. Then add purified water to a total volume of 3 mL, shake well, centrifuge for 10 min, and filter the supernatant to obtain the final product.

[0035] 4.2 HPLC detection method and chromatographic conditions Plotting the standard curve: Preparation of standard solutions: Weigh an appropriate amount of camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a 1 mg / mL standard stock solution; then serially dilute with methanol to obtain standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL, and 50.00 μg / mL. Plotting a standard curve: Inject the above standard solutions at concentrations from low to high, and plot the standard curve with concentration on the x-axis and peak area on the y-axis.

[0036] Detection method: 20 μL of Camptotheca acuminata fruit extract was analyzed by HPLC. The peak area obtained was used to calculate the corresponding concentration using a standard curve. The calculated concentration was then substituted into the following formula to calculate the camptothecin content in Camptotheca acuminata fruit.

[0037] C: Measured camptothecin concentration (μg / mL); V: Total volume of extract after dilution (mL); m sample : Sample weight of Camptotheca acuminata fruit (mg).

[0038] The chromatographic conditions are as follows: Chromatographic column: C18 Superb 5 μm, 4×150 mm (W); mobile phase: methanol-water (45 / 55.V / V), flow rate: 1 mL / min, injection volume: 20 μL; detection wavelength: 254 nm; column temperature: 30 °C.

[0039] 4.3 Optimization of Extraction Conditions 4.3.1 Content of seven-membered cucurbitacinar ring hypereutectic solvent (Q[7] / MA) The effect of Q[7] / MA content on extraction efficiency was investigated, with five Q[7] / MA contents of 5%, 15%, 25%, 35%, and 45% being examined. Figure 7 As shown in (a), with the increase of Q[7] / MA content, the peak area of ​​the extracted camptothecin also increases, and the peak area reaches the peak value when it reaches 25%.

[0040] 4.3.2 Material-to-liquid ratio like Figure 7 As shown in (b), the material-liquid ratio between Q[7] / MA and Camptotheca acuminata fruit was optimized. Six material-liquid ratios of 1:8, 1:12, 1:16, 1:20, 1:28 and 1:32 (g / mL) were investigated. When the material-liquid ratio was 1:28, the peak area of ​​camptothecin was maximized.

[0041] 4.3.3 Ultrasound time like Figure 7As shown in (c), the ultrasonic extraction time was optimized, and six ultrasonic times of 5 min, 10 min, 15 min, 20 min, 25 min and 30 min were examined. The results showed that the peak area increased with the increase of time, and the peak area was the largest at 15 min.

[0042] 4.3.4 Extraction Temperature The extraction temperature was investigated, with six different extraction temperatures set at 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃. Figure 7 As shown in (d), the peak area increases with increasing temperature, reaching its peak at 40℃.

[0043] 4.4 Measurement Results Therefore, the optimal extraction process is as follows: First, the Camptotheca acuminata fruit was dried in an oven at 50℃ for 8 hours. After drying, it was pulverized in a grinder and then passed through a 40-mesh sieve for later use. 250 μL of a seven-membered cucurbita supramolecular cocrystal solvent was added to 750 μL of purified water to obtain a 25% concentration of the seven-membered cucurbita supramolecular cocrystal solvent. Then, 50 mg of Camptotheca acuminata fruit was accurately weighed and added to the 25% concentration of the seven-membered cucurbita supramolecular cocrystal solvent. The mixture was ultrasonically extracted at 42℃ for 15 min, followed by centrifugation for 10 min to separate the Camptotheca acuminata fruit residue and the supramolecular cocrystal solvent. Purified water was then added to a total volume of 3 mL, and the mixture was shaken well and centrifuged for 10 min to obtain the Camptotheca acuminata fruit extract.

[0044] The camptothecin content in the camptothecin extract prepared using the optimal extraction process was determined to be 1.22 μg / mg. Furthermore, the recovery and reproducibility of the method were validated, and the results are shown in Table 1, confirming that the method has good accuracy and reproducibility.

[0045] Table 1. Results of Precision and Recovery Experiments 4.5. Comparison with traditional extraction methods To verify the extraction effect of the prepared seven-membered cucurbita supramolecular cocrystal solvent (Q[7] / MA), citric acid and malic acid were separately prepared into a citric acid-malic acid cocrystal solvent (CA / MA). The preparation method of CA / MA was as follows: 3.0g of citric acid, 2.0g of malic acid and 8.0mL of water were mixed in a large glass bottle and heated in an oil bath at 50°C with magnetic stirring at 400rpm for 1.5h. The resulting transparent liquid solvent was the CA / MA cocrystal solvent. The extraction conditions were the same as those for the Q[7] / MA solvent.

[0046] The extraction effects of Q[7] / MA, CA / MA, and 75% methanol (containing 1% HCl V / V) on Camptotheca acuminata fruit under the same extraction conditions were compared. The results are as follows: Figure 8 As shown, the extraction efficiency of the self-made supramolecular solvent is 3 times that of CA / MA and 2 times that of methanol solvent extraction, and the extraction time can reach equilibrium in 10 minutes, while the other two extraction methods require more than 30 minutes.

[0047] 4.6 Investigation of the anticancer activity of eutectic solvent on Camptotheca acuminata fruit extract Camptothecin, a component of Camptotheca acuminata fruit, has excellent anticancer activity. The anticancer activity of camptothecin extracted from Camptotheca acuminata fruit was tested using a seven-membered cucurbitacin supramolecular co-crystal solvent (Q[7] / MA). The results are as follows: Figure 9 , Figure 10 As shown. From Figure 9 As can be seen, the viability of 4T1 cells decreased in a concentration-dependent manner as the camptothecin concentration increased from 0 μM to 8 μM. Further fitting of cell viability with the logarithm of camptothecin concentration yielded a dose-response curve. Figure 10 The half-maximal inhibitory concentration (IC50) of camptothecin on 4T1 cells was calculated. 50 The concentration was 5.401 μM, indicating that camptothecin has significant in vitro inhibitory activity against the proliferation of 4T1 cells, and the inhibitory effect is positively correlated with the concentration.

[0048] In summary, this invention prepares a supramolecular cocrystal solvent for seven-membered cucurbitacin-malic acid. The viscosity and density of this solvent decrease with increasing temperature. This solvent is a green extraction solvent that does not use organic solvents when extracting active ingredients from plants, while the extracted active ingredients retain their intrinsic activity. The preparation method of the supramolecular cocrystal solvent is simple, with high extraction efficiency, and can utilize the molecular recognition ability of the seven-membered cucurbitacin to achieve efficient and green extraction. Compared with traditional organic solvent extraction processes, it has significant advantages such as high extraction rate, environmental friendliness, and ease of operation.

Claims

1. A method for preparing a seven-membered cucurbitacinoid supramolecular eutectic solvent, characterized in that: Malic acid is used as a hydrogen bond donor and cucurbitacinium as a hydrogen bond acceptor. Malic acid and cucurbitacinium are weighed and mixed in a mass ratio of 8-12:

1. Then, water is added to the mixture, which is 25-35% of the total mass of malic acid and cucurbitacinium. The mixture is heated and stirred at 80-100℃ for 30-50 minutes to obtain a homogeneous and transparent solution, which is the supramolecular eutectic solvent of cucurbitacinium.

2. The method for preparing the seven-membered cucurbitacinoid supramolecular eutectic solvent according to claim 1, characterized in that: Malic acid is used as a hydrogen bond donor and cucurbitacinium as a hydrogen bond acceptor. Malic acid and cucurbitacinium are weighed and mixed in a mass ratio of 9-11:

1. Then, water is added to the mixture, which is 25-30% of the total mass of malic acid and cucurbitacinium. The mixture is heated and stirred at 85-95℃ for 35-45 minutes to obtain a homogeneous and transparent solution, which is the supramolecular eutectic solvent of cucurbitacinium.

3. The method for preparing the seven-membered cucurbitacinoid supramolecular eutectic solvent according to claim 2, characterized in that: Malic acid was used as a hydrogen bond donor and cucurbitacin was used as a hydrogen bond acceptor. Malic acid and cucurbitacin were weighed in a mass ratio of 10:1, mixed, and then water was added in a mixture equal to 25% of the total mass of malic acid and cucurbitacin. The mixture was heated and stirred at 90°C for 40 minutes to obtain a uniform and transparent solution, which is the supramolecular eutectic solvent of cucurbitacin.

4. The application of the seven-membered cucurbitacin supramolecular eutectic solvent according to any one of claims 1-3 in the determination of camptothecin content in Camptotheca acuminata fruit, characterized in that: Camptothecin was extracted from Camptotheca acuminata fruit using a seven-membered cucurbitacin supramolecular eutectic solvent, and the content of the extracted camptothecin was determined by HPLC.

5. The application according to claim 4, characterized in that: The content determination was performed according to the following steps: (1) Pretreatment of Camptotheca acuminata fruit: Place Camptotheca acuminata fruit in an oven at 45-55℃ and dry for 7-9 hours. After drying, put it into a pulverizer and pulverize it. Then pass it through a 30-60 mesh sieve to obtain Camptotheca acuminata fruit powder for later use. (2) Extraction of Camptotheca acuminata fruit: Accurately weigh 40-50 mg of Camptotheca acuminata fruit powder, add 250-450 μL of seven-membered cucurbita ring supramolecular eutectic solvent and 750-550 μL of purified water, and extract by ultrasonication at 30-45℃ for 5-30 min. Then add purified water to the total volume of 3 mL, shake evenly and centrifuge for 8-12 min. Take the supernatant and filter to obtain Camptotheca acuminata fruit extract. (3) Preparation of standard solutions: Weigh the camptothecin standard, add 10 μL of 12M HCl to aid dissolution, and dilute with methanol to prepare a standard stock solution with a concentration of 1 mg / mL; then dilute stepwise with methanol to prepare standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL, respectively, for later use; (4) Preparation of standard curve: Standard solutions with concentrations of 0.25 μg / mL, 5.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 25.00 μg / mL and 50.00 μg / mL were analyzed by HPLC at concentrations from low to high. The standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The chromatographic conditions were as follows: Chromatographic column: C18 Superb 5um, 4×150mm; mobile phase: methanol-water, methanol to water volume ratio: 45:55, flow rate: 1mL / min, injection volume: 20μL; detection wavelength: 254nm; column temperature: 30℃. (5) Content determination: Take the extract of Camptotheca acuminata fruit and perform HPLC analysis and detection. The chromatographic conditions are the same as in step (4). The obtained peak area is substituted into the standard curve to calculate the corresponding concentration.

6. The application according to claim 5, characterized in that: In step (1), the pretreatment of Camptotheca acuminata fruit is as follows: the Camptotheca acuminata fruit is placed in an oven at 48-52℃ and dried for 7.5-8.5 hours. After drying, it is crushed in a pulverizer and then passed through a 40-50 mesh sieve to obtain Camptotheca acuminata fruit powder for later use.

7. The application according to claim 6, characterized in that: In step (1), the pretreatment of Camptotheca acuminata fruit is as follows: the Camptotheca acuminata fruit is placed in an oven at 50°C and dried for 8 hours. After drying, it is placed in a pulverizer and pulverized. Then it is passed through a 40-mesh sieve to obtain Camptotheca acuminata fruit powder for later use.

8. The application according to claim 5, characterized in that: In step (2), the camptotheca fruit is extracted: accurately weigh 50-65 mg of camptotheca fruit powder, add 250-350 μL of seven-membered cucurbita supramolecular eutectic solvent and 750-650 μL of purified water, and extract by ultrasonication at 40-45℃ for 5-15 min. Then add purified water to a total volume of 3 mL, shake evenly and centrifuge for 9-11 min. Take the supernatant and filter to obtain the camptotheca fruit extract.

9. The application according to claim 8, characterized in that: In step (2), the camptotheca fruit is extracted: accurately weigh 50 mg of camptotheca fruit powder, add 250 μL of seven-membered cucurbita supramolecular eutectic solvent and 750 μL of purified water, and extract by ultrasonication at 40°C for 15 min. Then add purified water to a total volume of 3 mL, shake evenly and centrifuge for 10 min, take the supernatant and filter to obtain the camptotheca fruit extract.

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