Preparation method and application of inclusion compound for improving stability of strigolactones
By encapsulating GR24 within the voids of the host molecule using inclusion complex technology, the instability problem of strigolactones was solved, and their stability and water solubility under acidic and alkaline conditions were improved, thus promoting increased bioavailability.
Patent Information
- Application Number
- CN202511995169.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-01-30
AI Technical Summary
The poor water solubility and unstable molecular structure of strigolactones limit their application under acidic and alkaline conditions.
Using inclusion complex technology, GR24 is embedded in the cavity structure of host molecules such as cyclodextrin or its derivatives, crown ethers, and calixarenes. A stable inclusion complex is formed by preparing a GR24 inclusion complex solution and then performing rotary evaporation and freeze-drying.
It significantly improved the acid, alkali and thermal stability of strigolactone, enhanced its solubility in water, extended the product shelf life and improved bioavailability.
Smart Images
Figure CN121420992A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product chemistry, specifically relating to a method for preparing inclusion compounds that improve the stability of strigolactones and their application. Background Technology
[0002] Strigolactones (SLs) are a class of sesquiterpenoids derived from carotenoids. Typical SLs consist of a tricyclic lactone (ABC tricyclic ring) coupled to an α,β-unsaturated furan ring (D ring) via an enol ether bond (Formula I). GR24 (Formula II), GR7 (Formula III), and 7FGR24 (Formula IV) are three synthetically produced SL analogs, widely used as standards for SL biosynthesis and signal transduction experiments. As the seventh major class of novel plant hormones, SLs play a crucial role in regulating plant environmental adaptation and growth, such as inhibiting lateral bud germination, adjusting crop architecture, promoting symbiotic relationships between plants and soil microorganisms, and enhancing host resistance. Furthermore, SLs can inhibit breast cancer cell proliferation and induce apoptosis. However, SLs have extremely poor water solubility and unstable enol ether and lactone bonds in their molecular structure, easily decomposing under acidic and alkaline conditions, which greatly limits their practical applications.
[0003]
[0004]
[0005] Inclusion molecule technology, by encapsulating guest molecules within the voids of host molecules to form inclusion compounds, can effectively improve the stability of guest molecules, increase their solubility, regulate drug dissolution rates, and enhance bioavailability. Although significant progress has been made in improving the stability of natural active molecules using inclusion molecule technology, research on improving the stability of strigolactones has not yet been reported. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing and applying inclusion compounds that improve the stability of strigolactones.
[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0008] A method for preparing an inclusion complex that improves the stability of strigolactones includes the following steps:
[0009] Step 1: Preparation of GR24 inclusion complex solution:
[0010] The host molecule was dissolved in deionized water to prepare a GR24 solution. The GR24 solution was then slowly added dropwise to the aqueous solution of the host molecule, and the mixture was stirred for a certain period of time to obtain a GR24 inclusion complex solution.
[0011] Step 2: Preparation of GR24 inclusion complex:
[0012] The obtained GR24 inclusion complex solution was filtered through a hydrophilic filter membrane, and then the mixed solution was concentrated by rotary evaporation. Finally, the concentrated solution was freeze-dried to obtain the GR24 inclusion complex.
[0013] In step 1, the ratio of the main molecule, GR24, and deionized water is (0.1567 ~ 15.67) g : (0.030 ~ 3.0) g : (23.51 ~ 2351) mL; the reaction temperature is 15 ~ 50℃, and the reaction time is 6 ~ 48 h.
[0014] In step 1, the concentration of the GR24 solution is 10. -6 mol / L~10 -2 mol / L.
[0015] In step 1, the main molecules include cyclodextrin (α-cyclodextrin (CD) or its derivatives, γ-cyclodextrin or its derivatives, β-cyclodextrin or its derivatives (such as hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, 2-methyl-β-cyclodextrin, 3-methyl-β-cyclodextrin), amino-cyclodextrin, sulfobutyl-β-cyclodextrin, etc.), crown ethers, calixarenes, columnar aromatics (diethoxy columnar[6]arene, columnar[6]arene, columnar[7]arene, columnar[9]arene, columnar
[10] arene) or cucurbita (cucurbita[7]urene, cucurbita[8]urene, cucurbita
[10] urene), etc.
[0016] In step 1, the solvent of the GR24 solution is methanol, ethanol, acetone, acetonitrile, or dimethyl sulfoxide, etc.
[0017] In step 1, the reaction time is 6–48 h and the reaction temperature is 15–50 ℃.
[0018] In step 2, the rotary evaporation temperature is 30 ~ 100 ℃, the rotary evaporation time is 10 ~ 60 min, and the rotation speed is 50 ~ 100 rpm; the filter membrane pore size is 0.22 ~ 0.45 μm, the freeze-drying temperature is -18 ~ -40 ℃, and the drying time is 24 ~ 72 h.
[0019] Furthermore, the inclusion complex prepared by this invention can enhance the acid, basic and thermal stability of strigolactones and improve their solubility in water, which can help develop new strigolactone pesticide formulations and improve bioavailability.
[0020] The beneficial effects of this invention are as follows:
[0021] 1) This invention innovatively uses inclusion technology to coat strigolactones. The experimental method is simple to operate, the coating effect is good, and it significantly improves the acid, alkali and thermal stability and water solubility of strigolactones, thereby helping to improve the bioavailability of strigolactones and accelerate their promotion and application.
[0022] 2) The present invention uses freeze-drying to prepare GR24-containing inclusion complexes. The resulting product is very stable and has a long shelf life of more than 6 months at room temperature and in a dry environment, providing a new method for the preservation of strigolactones. Attached Figure Description
[0023] Figure 1 These are scanning electron microscope images of β-CD, GR24, a physical mixture of β-CD and GR24, and a β-CD / GR24 inclusion complex from Test Example 1 of the present invention.
[0024] Figure 2 The infrared spectra of β-CD, GR24, the physical mixture of β-CD and GR24, and the β-CD / GR24 inclusion complex in Test Example 1 of this invention are shown.
[0025] Figure 3 This is an X-ray diffraction pattern of β-CD, GR24, a physical mixture of β-CD and GR24, and a β-CD / GR24 inclusion complex in Test Example 1 of the present invention;
[0026] Figure 4 Differential scanning calorimetry (DSC) images of β-CD, GR24, physical mixtures of β-CD and GR24, and β-CD / GR24 inclusion complex in Test Example 1 of this invention.
[0027] Figure 5 Thermogravimetric curves of β-CD, GR24, physical mixtures of β-CD and GR24, and β-CD / GR24 inclusion complex in Test Example 1 of this invention are shown.
[0028] Figure 6 This is the Zeta potential diagram of GR24 and β-CD / GR24 inclusion complex in Test Example 1 of this invention;
[0029] Figure 7 This is a high-performance liquid chromatogram of GR24 after treatment with acidic and alkaline solutions with pH values of 1, 7 and 13 in Test Example 1 of the present invention.
[0030] Figure 8 This is a high-performance liquid chromatography (HPLC) chromatogram of the β-CD / GR24 inclusion complex in Test Example 1 of the present invention after treatment with acidic and alkaline solutions at pH 1, 7 and 13, respectively.
[0031] Figure 9 This is a graph showing the change in the mass fraction of GR24 in the composition of GR24 and β-CD / GR24 inclusion complex in Test Example 1 of the present invention after treatment with acidic and alkaline solutions at pH values of 1, 7 and 13, respectively, as a function of treatment time.
[0032] Figure 10 This is an electron microscope image showing the results of the experiment on the "suicidal" germination activity of broomrape seeds induced by GR24 and β-CD / GR24 inclusion complex in Test Example 1 of this invention. Detailed Implementation
[0033] In this document, to enable readers to better understand and recognize the solutions of the present invention, specific implementation methods and effects of the present invention will be illustrated through the following examples. Furthermore, the endpoints of the ranges and any numerical values presented herein are not limited to such precise ranges or values.
[0034] Example 1
[0035] A method for preparing an inclusion complex that improves the stability of strigolactones includes the following steps:
[0036] (a) Preparation of β-CD inclusion complex solution:
[0037] 30 mg of GR24 was weighed and added to 1.5 mL of anhydrous ethanol, and sonicated for 30 min to dissolve it rapidly. Then, 0.1338 g of β-CD was weighed and added to 20.62 mL of ultrapure water, heated at 70 °C and magnetically stirred to completely dissolve the β-CD. Subsequently, 1.5 mL of the GR24 solution was slowly added to 20.62 mL of the β-CD aqueous solution, and the resulting mixture was stirred at room temperature for 24 h using a magnetic stirrer to obtain a β-CD inclusion complex solution.
[0038] (b) Preparation of β-CD inclusion complex:
[0039] The inclusion complex solution obtained in (a) was filtered through a 0.45 μm hydrophilic microporous membrane, then rotary evaporated at 50 °C and 100 rpm for 20 min, and finally freeze-dried for 24 h to obtain a pale yellow β-CD / GR24 inclusion complex powder.
[0040] Examples 2-4
[0041] The preparation methods of Examples 2-4 are the same as those of Example 1, except that the mass of GR24 added in step (a) is 15 mg, 45 mg and 60 mg respectively, and the corresponding volumes of anhydrous ethanol are 0.75 mL, 2.25 mL and 3.00 mL respectively.
[0042] Examples 5-6
[0043] The preparation methods of Examples 5 and 6 are the same as those of Example 1, except that GR24 in step (a) is replaced with equimolar amounts of 7FGR24 and GR7.
[0044] Examples 7-9
[0045] The preparation methods of Examples 7-9 are the same as those of Example 1, except that β-CD in step (a) is replaced with equimolar amounts of 2-hydroxypropyl-β-CD, 3-methyl-β-CD, and diethoxy aromatic hydrocarbons.
[0046] Examples 10-11
[0047] The preparation methods of Examples 10 and 11 are the same as those of Example 1, except that the reaction temperatures in step (a) are 15 °C and 50 °C, respectively.
[0048] Experimental data description
[0049] The surface microstructure of the β-CD / GR24 inclusion complex prepared in Example 1 is shown in the figure. Figure 1 .like Figure 1 As shown, the small molecule GR24 is in the form of random particles, while β-CD has an irregular blocky structure with some cracks on the surface. The microstructure of the β-CD / GR24 mixture is similar to that of β-CD, except that its surface is covered with a layer of fine particles, namely GR24. In contrast, the β-CD / GR24 inclusion complex has a smooth, plate-like structure with no particles adhering to its surface, indicating that GR24 has entered the cavity structure of β-CD, thus proving the formation of the inclusion complex.
[0050] The infrared spectrum of the β-CD / GR24 inclusion complex prepared in Example 1 is shown in Figure 1. Figure 2 As the results show, 3098.5 cm -1 The characteristic absorption peak at 1726.5 cm⁻¹ corresponds to the CH stretching vibration peak on the aromatic ring of GR24. -1 The characteristic absorption peak at 1171.7 cm⁻¹ corresponds to the C=O stretching vibration peak on the ester group. -1 and 1003.5 cm -1 The absorption peaks around 1670.0 cm⁻¹ are characteristic absorption peaks of enol ether bonds (C=CO). -1 The absorption peak at the wavenumber is the characteristic absorption peak of C=C on the D ring. 1640.0 cm⁻¹ -1The peak at 741.7 cm⁻¹ is the characteristic absorption peak of C=C on ring A. -1 The peak at 3278.4 cm⁻¹ corresponds to the CH bending vibration of the benzene ring. In the FT-TR spectrum of β-CD, this peak is located at 3278.4 cm⁻¹. -1 The broad peak at 2926.2 cm⁻¹ is a characteristic absorption peak of the OH stretching vibration. -1 The absorption peak in the wavenumber range is the CH stretching vibration peak. (1330 cm⁻¹) -1 The peak at this location is the CO bending vibration peak. 1149.9 cm⁻¹ -1 and 1019.1 cm -1 The absorption peak at 1726.5 cm⁻¹ is attributed to the stretching vibration of COC. The FT-TR spectrum of a physical mixture of β-CD and GR24 can be viewed as a superposition of the β-CD and GR24 spectra, as shown at 1726.5 cm⁻¹. -1 The C=O stretching vibration peak is still present. The FT-TR spectrum of the β-CD / GR24 inclusion complex has a peak shape almost identical to that of β-CD, ranging from 1300 to 1500 cm⁻¹. -1 There is some disappearance of small peaks within the range, 1726.5 cm. -1 The C=O stretching vibration peak shifted to the high-frequency region at 1732.7 cm⁻¹. -1 This indicates that hydrogen bonds are formed between β-CD and GR24, resulting in an inclusion complex.
[0051] The crystalline structures of GR24, β-CD, β-CD / GR24 physical mixtures, and β-CD / GR24 inclusion complexes were analyzed using X-ray diffraction. Figure 3 As shown, GR24 exhibits sharp, strong diffraction peaks at 6.42°, 12.80°, 13.74°, 15.64°, 23.26°, and 25.42°, indicating that GR24 possesses a specific crystal structure. β-CD also exhibits sharp, strong peaks at 9.12°, 10.76°, 12.6°, and 27.12°, indicating that it also possesses a crystalline structure. The XRD pattern of the β-CD / GR24 physical mixture is a simple superposition of the β-CD and GR24 crystal structures. In comparison, the crystalline peaks of the β-CD / GR24 inclusion complex are reduced, and the peak intensities are significantly weakened, indicating a change in the crystalline structure. This may be because GR24 enters the cavities of β-CD and is thus dispersed by β-CD, leading to disorder in the inclusion complex crystal and resulting in weakened diffraction peak intensities.
[0052] Differential scanning calorimetry (DSC) plots of GR24, β-CD, β-CD / GR24 physical mixtures, and β-CD / GR24 inclusion complexes are shown below. Figure 4The melting points of β-CD and GR24 are approximately 146 °C and 154 °C, respectively. The DSC curve of the β-CD / GR24 physical mixture shows a superposition of β-CD and GR24, with characteristic endothermic peaks of β-CD and GR24 appearing at 139 °C and 155 °C, respectively. The β-CD / GR24 inclusion complex, however, exhibits a broad endothermic peak at a lower temperature of 127 °C and a small endothermic peak at a higher temperature of 155 °C. The melting peak of GR24 essentially disappears, indicating that GR24 has been largely included by β-CD, forming a new crystal structure, consistent with the XRD results.
[0053] Thermogravimetric analysis of GR24, β-CD, β-CD / GR24 physical mixtures and β-CD / GR24 inclusion complexes ( Figure 5 (Left figure) and its first-order differential curve ( Figure 5 (See right figure) Results Figure 5 As shown in the figure, GR24 exhibits a relatively high initial decomposition temperature of 252.5 °C. β-CD, due to the presence of numerous hydrophilic hydroxyl groups on its surface, binds a significant amount of free and crystalline water, resulting in slight decomposition before 200 °C. The initial decomposition temperatures of the physical mixture and inclusion complex are similar to those of β-CD, but the decomposition rates are lower. The first-order differential thermogravimetric curves reveal that the maximum decomposition temperatures of the β-CD / GR24 inclusion complex are significantly higher than those of GR24, at 310.8 °C and 274.1 °C respectively, indicating enhanced thermal stability of the inclusion complex.
[0054] The zeta potential analysis results of GR24 and β-CD / GR24 inclusion complex dispersed in aqueous solution are shown in the figure. Figure 6 The zeta potential of the β-CD / GR24 inclusion complex is significantly higher than that of the guest molecule GR24, indicating that there is a stronger charge repulsion between the host and guest molecules in the inclusion complex, which helps to maintain the stability of the inclusion complex structure in water and improves the solubility and stability of GR24 in aqueous solution.
[0055] The stability of GR24 and β-CD / GR24 inclusion complex under acidic and alkaline solutions at pH 1, 7, and 13 was then investigated using high performance liquid chromatography. Figure 7 , Figure 8 ), and compiled the curves showing the change in the mass fraction of GR24 with processing time ( Figure 9 ).like Figure 7As shown, the elution time of the characteristic peak of GR24 is around 7.8 min. After treatment with a neutral solution at pH 7 for 48 h, the mass of GR24 remains essentially unchanged, maintaining 99.78% of its initial mass, with the characteristic peaks around 6.4 min and 0.4 min being solvent absorption peaks. However, after treatment with a strong acid solution at pH 1 for a period of time, the peak intensity of the GR24 characteristic peak significantly weakens, attributed to partial decomposition of GR24. In addition, the position of the GR24 characteristic peak shifts, appearing around 8.0 min, due to changes in the interaction between GR24 and the solvent caused by the altered solution environment. After treatment with a strong alkaline solution at pH 13 for a period of time, two new absorption peaks appear at 4.2 min and 5.6 min, and the characteristic absorption peak of GR24 disappears, indicating complete decomposition of GR24. After treatment with strong acid and strong alkaline solutions at pH 1 and 13 for 48 h, GR24 decomposed by 27.7% and 100%, respectively. When GR24 was encapsulated with β-CD, the decomposition rate of GR24 in the encapsulation solution was significantly reduced to 14.7% and 31.1% after treatment with strong acid and strong base solutions at pH 1 and 13 for 48 h, respectively, indicating that the acid and base stability of GR24 was significantly improved.
[0056] Test Example 1
[0057] Using sunflower broomrape seeds as the test subject, and 0.1 wt% dimethyl sulfoxide aqueous solution as the seed germination stock solution, the effect of the β-CD / GR24 inclusion complex on the bioactivity of GR24 was tested through a seed germination induction experiment of parasitic plants. Figure 10 As shown, the mother liquor did not induce germination of sunflower broomrape seeds, with a germination rate of 0%. The main molecule β-CD had little effect on the germination of parasitic plant seeds, with a germination rate of only 3.2 ± 2.5%. The germination rate of sunflower broomrape seeds induced by GR24 was 38.1 ± 5.3%, while the germination rate induced by the β-CD / GR24 inclusion complex was almost the same as that of GR24, at 34.8 ± 5.6%. This indicates that the inclusion complex has a very low effect on the biological activity of GR24, but can significantly improve its stability and solubility.
Claims
1. A process for the preparation of a clathrate for improving stability of jolkinolide, characterized by: The method comprises the following steps: Step 1: Preparation of GR24 inclusion complex solution The host molecule is dissolved in deionized water, and a GR24 solution is prepared, then the GR24 solution is slowly added to the host molecule aqueous solution, stirred for a certain time to obtain a GR24 inclusion complex solution; Step 2: Preparation of GR24 inclusion complex The obtained GR24 inclusion complex solution is filtered with a hydrophilic filter membrane, then the mixed solution is concentrated by rotary evaporation, and finally the concentrated solution is freeze-dried to obtain the GR24 inclusion complex.
2. The production method according to claim 1, characterized by The dosage ratio of the host molecule, GR24 and deionized water is (0.1567 ~ 15.67) g: (0.030 ~ 3.0) g: (23.51 ~ 2351) mL; the reaction temperature is 15 ~ 50℃, and the reaction time is 6 ~ 48 h.
3. The production method according to claim 1, wherein The host molecule includes any one of cyclodextrin, crown ether, calixarene, pillararene or cucurbituril.
4. The production method according to claim 1, wherein The concentration of the GR24 solution in step 1 is 10 -6 mol / L to 10 -2 mol / L.
5. The production method according to claim 1, wherein In step 1, the solvent of the GR24 solution is methanol, ethanol, acetone, acetonitrile or dimethyl sulfoxide.
6. The production method according to claim 1, wherein In step 1, the reaction time is 6 ~ 48 h, and the reaction temperature is 15 ~ 50℃.
7. The production method according to claim 1, wherein In step 2, the rotary evaporation temperature is 30 ~ 100℃, the rotary evaporation time is 10 ~ 60 min, and the rotation speed is 50 ~ 100 rpm.
8. The production method according to claim 1, wherein In step 2, the filter membrane pore size is 0.22 ~ 0.45μm.
9. The production method according to claim 1, wherein In step 2, the freeze-drying temperature is -18 ~ -40℃, and the drying time is 24 ~ 72 h.
10. The inclusion complex prepared by the preparation method of any one of claims 1 ~ 9 is used to improve the stability and solubility of strigolactone, which is helpful for the development of new pesticide preparation products of strigolactone, and improves the bioavailability.