An adrenaline-flavonoid carbon glycoside supramolecular complex preparation method using acoustic resonance mixing technology

Abstract

The present application relates to the technical field of pharmaceutical preparation, in particular to a preparation method of an adrenaline-flavone carbon glycoside supramolecular complex by using acoustic resonance mixing technology, comprising: uniformly mixing adrenaline and flavone carbon glycoside at a molar ratio of 1:0.5-1:3, placing them in a resonance cavity of an acoustic resonance mixer, adding grinding medium, and performing resonance mixing treatment at a resonance frequency of 10-40 Hz for 5-120 min; and collecting the complex powder after the treatment to obtain the adrenaline-flavone carbon glycoside supramolecular complex. The adrenaline-flavone carbon glycoside supramolecular complex prepared by the present application has excellent stability and good dissolution and release characteristics, can effectively solve the stability problem of adrenaline during storage and use, and can simultaneously exert the antioxidant and anti-inflammatory effects of flavone carbon glycoside, thus having important clinical application value and market application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a method for preparing an adrenaline-flavonoid C-glycoside supramolecular complex using acoustic resonance mixing technology. Background Technology

[0002] Epinephrine is a catecholamine hormone secreted by the adrenal medulla and is one of the most important emergency drugs in clinical practice. Its main pharmacological effects include: activating α-receptors to cause vasoconstriction and increased blood pressure; activating β-receptors to enhance myocardial contractility and increase heart rate; and activating β2-receptors to dilate bronchial smooth muscle. Based on these pharmacological effects, epinephrine is widely used in the rescue and treatment of critical illnesses such as anaphylactic shock, cardiac arrest, and acute exacerbations of bronchial asthma. However, the chemical structure of epinephrine determines its extremely unstable nature. The epinephrine molecule contains catechol and β-hydroxylamine structures, and these functional groups make it highly susceptible to degradation by the following factors: 1) The catechol structure is easily oxidized by oxygen in the air to produce adrenochrome, which further polymerizes to form a brown polymer, leading to loss of efficacy and increased toxicity.

[0003] 2) Light can catalyze the oxidation of adrenaline, accelerating its decomposition.

[0004] 3) Adrenaline is unstable under both acidic and alkaline conditions and is prone to racemization, which converts the pharmacologically active R-configuration into the inactive S-configuration.

[0005] 4) Increased temperature significantly accelerates the degradation of adrenaline. These conditions make the use and storage of adrenaline and its preparations difficult. Currently, commercially available adrenaline injections typically require the addition of antioxidants (such as sodium bisulfite) and metal ion chelating agents (such as EDTA), and must be stored at low temperatures and protected from light. Even so, the shelf life of adrenaline injections is usually only 1-2 years, and they must be used immediately after opening. This instability causes significant inconvenience in clinical emergency care, especially in scenarios lacking cold chain conditions such as pre-hospital emergency care and battlefield medical care. Adrenaline failure may result in patients missing precious opportunities for rescue.

[0006] Flavonoid C-glycosides are an important class of natural products, widely found in medicinal plants. Unlike ordinary O-glycosides, the glycosyl group of flavonoid C-glycosides is directly linked to the flavonoid nucleus via a C-C bond. This structure endows them with unique stability—the C-C bond is highly resistant to acid, alkali, and enzymatic hydrolysis, allowing them to maintain structural integrity in both in vivo and in vitro environments. Orientin and isoorientin are two representative flavonoid C-glycosides, namely luteolin-8-C-glucoside and luteolin-6-C-glucoside, respectively. These two compounds exhibit significant antioxidant activity. Their benzene ring contains a catechol structure (3',4'-dihydroxy) similar to that of adrenaline, enabling them to exert antioxidant effects through the following mechanisms: Direct free radical scavenging: The catechol structure provides hydrogen atoms to react with free radicals, terminating free radical chain reactions. Metal ion chelation: The flavonoid structure can chelate with transition metal ions (such as Fe²⁺). + Cu² + It chelates and inhibits hydroxyl radicals generated by the Fenton reaction. It activates the endogenous antioxidant system: it can upregulate the expression of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Furthermore, arbutin and isohypoglycine also possess significant anti-inflammatory activity, inhibiting the activation of the NF-κB signaling pathway and reducing the production of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This anti-inflammatory effect aligns perfectly with the emergency application of adrenaline—in the treatment of anaphylactic shock, in addition to the vasoactive effects of adrenaline, controlling excessive inflammatory responses is equally crucial.

[0007] Resonance Acoustic Mixing (RAM) is an emerging solid-state processing technology. Its principle involves using a resonant frequency of 10–40 Hz to drive the grinding media to resonate, thereby generating high-intensity mechanical forces between solid materials. Compared to traditional ball milling, RAM offers several unique advantages: acoustic resonance primarily relies on the vibration and collision of the media to generate mechanical forces, rather than the shearing and friction of traditional ball milling, which helps protect the activity of heat-sensitive substances; energy transfer is more efficient in the resonant state, enabling thorough mixing and ultrafine pulverization of materials in a shorter time; furthermore, the use of non-metallic grinding media such as zirconium oxide avoids contamination of the product by metal debris, and the entire process requires no organic solvents, conforming to green chemistry principles. Currently, RAM has been successfully applied in the extraction of natural products, the preparation of nanomaterials, and the preparation of drug solid dispersions. Studies have shown that acoustic resonance treatment can disrupt plant cell walls, promoting the release of active ingredients; simultaneously, under the action of mechanical forces, supramolecular interactions can form between drug molecules and carrier materials, improving drug solubility and stability.

[0008] Although existing research has explored the compatibility of adrenaline with various excipients, current technologies still have the following shortcomings: Traditional antioxidants (such as sodium bisulfite), while able to delay the oxidation of adrenaline, may themselves cause allergic reactions and cannot fundamentally solve the stability problem of adrenaline. Most existing adrenaline preparations are simple physical mixtures with a lack of molecular-level interactions between components, resulting in limited synergistic effects. No research reports have yet described a technical solution utilizing flavonoid C-glycosides to form supramolecular complexes with adrenaline to improve its stability. Existing mechanochemical preparation methods often employ high-energy ball milling, which may lead to the degradation of heat-sensitive drugs. Therefore, developing a new technology that can effectively solve the stability problem of adrenaline while simultaneously leveraging the synergistic antioxidant and anti-inflammatory effects of flavonoid C-glycosides has significant clinical application value and market potential. Summary of the Invention

[0009] In view of this, the present invention proposes a method for preparing adrenaline-flavonoid C-glycoside supramolecular complexes using acoustic resonance mixing technology. Based on the rational design of complementary molecular structures, this invention opens up a new direction for the development of adrenaline preparations. The adrenaline-flavonoid C-glycosides prepared by this method exhibit excellent stability, antioxidant activity, and synergistic anti-inflammatory effects.

[0010] The specific technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a method for preparing an adrenaline-flavonoid carbon glycoside supramolecular complex using acoustic resonance mixing technology, comprising: mixing adrenaline and flavonoid carbon glycosides uniformly at a molar ratio of 1:0.5 to 1:3, placing the mixture in the resonance cavity of an acoustic resonance mixer, adding grinding media, performing resonance mixing treatment at a resonance frequency of 10 to 40 Hz for 5 to 120 min, collecting the complex powder after the treatment, and obtaining the adrenaline-flavonoid carbon glycoside supramolecular complex.

[0011] Furthermore, the adrenaline is pharmaceutical grade adrenaline.

[0012] Furthermore, the flavonoid glycoside is selected from one of the following: pursinoside, isopursinoside, puerarin, vitexin, and isovitexin.

[0013] Furthermore, the flavonoid C-glycoside is preferably isopropanol.

[0014] Furthermore, the molar ratio of adrenaline to flavonoid C-glycoside is preferably 1:1.

[0015] Furthermore, the resonant cavity material of the acoustic resonance mixer is selected from polytetrafluoroethylene or stainless steel, and its volume is 100~1000 mL.

[0016] Furthermore, the resonant cavity material of the acoustic resonance mixer is preferably polytetrafluoroethylene (PTFE), and the volume of the PTFE acoustic resonance mixer is preferably 600 mL.

[0017] Furthermore, the grinding media is a zirconia ball, agate ball, or ceramic ball with a diameter of 10-25 mm.

[0018] Furthermore, the grinding medium is preferably a zirconia ball, and the diameter of the zirconia ball is preferably 15 mm.

[0019] Furthermore, the mass ratio of the grinding media to the material is 1:1 to 20:1.

[0020] Furthermore, the mass ratio of the grinding media to the material is preferably 10:1.

[0021] Furthermore, the resonant frequency is preferably 30 Hz.

[0022] Furthermore, the resonant mixing time is preferably 30 min.

[0023] Furthermore, the adrenaline-flavonoid carbon glycoside supramolecular complex has a particle size in the nanometer range.

[0024] Secondly, the present invention provides the use of the adrenaline-flavonoid carbon glycoside supramolecular complex prepared by the above method in the preparation of drugs for treating anaphylactic shock and / or allergic asthma.

[0025] Furthermore, the drug has synergistic anti-inflammatory effects.

[0026] Furthermore, the drug is an injection or a powder.

[0027] Furthermore, the drug is administered via injection.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improves the stability of adrenaline: The catechol structure of flavonoid C-glycosides can act as a "sacrificial antioxidant" and be preferentially oxidized, thereby protecting the phenolic hydroxyl group of adrenaline. At the same time, the hydrogen bond network in the supramolecular complex can stabilize the chiral center of adrenaline and inhibit its racemization.

[0029] (2) Synergistic anti-inflammatory effect: Flavonoid C-glycosides themselves have the effect of inhibiting the NF-κB pathway and reducing the production of inflammatory factors. When they form a supramolecular complex with adrenaline, the two can work synergistically on multiple pathological aspects of anaphylactic shock: adrenaline rapidly reverses vasodilation and bronchospasm, while flavonoid C-glycosides inhibit excessive inflammatory response, thereby achieving a more comprehensive therapeutic effect.

[0030] (3) Improve drug solubility and release characteristics: Acoustic resonance mixing treatment pulverizes the drug complex to the molecular level, which significantly increases the specific surface area and thus improves the dissolution rate. Then, through redispersing and homogenization, intermolecular chemical interactions occur, thereby improving the drug dissolution rate and meeting the requirements of rapid onset of action for emergency drugs.

[0031] (4) The preparation process is green and environmentally friendly: the preparation process of the present invention is carried out entirely under solid conditions, without the use of organic solvents, and there is no problem of solvent residue; the acoustic resonance mixing temperature is low, which will not lead to the degradation of heat-sensitive drugs; the process is simple, the production cycle is short, and it is easy to scale up to industrial scale. Attached Figure Description

[0032] Figure 1 The effect of the molar ratio of adrenaline to isorhamnetin on the solubility of adrenaline.

[0033] Figure 2 The effect of RAM processing frequency on adrenaline solubility and retention rate is shown; the blue line represents adrenaline solubility, and the yellow line represents adrenaline retention rate.

[0034] Figure 3 The effect of RAM processing time on adrenaline solubility and retention rate is shown; the blue line represents adrenaline solubility, and the yellow line represents adrenaline retention rate.

[0035] Figure 4 X-ray powder diffraction patterns of adrenaline, isorhamnetin and their complexes.

[0036] Figure 5 This is a comparison of the infrared spectra of adrenaline, isohypoglycinin and their complexes.

[0037] Figure 6 Differential scanning calorimetry (DSC) curves of the adrenaline-isoharonin complex and the raw materials.

[0038] Figure 7 The nanoparticle size of the adrenaline-isoharonin complex is [not specified].

[0039] Figure 8 The curves show the content changes of the adrenaline-isoharonin complex and the active pharmaceutical ingredient during accelerated stability testing.

[0040] Figure 9 The effect of adrenaline-isoharonin complex on the survival rate of mice with anaphylactic shock.

[0041] Figure 10 The figures show the total cell count and eosinophil count of mice after different experimental treatments. The gray bars represent the total cell count of mice, and the white bars represent the eosinophil count.

[0042] Figure 11 The total serum IgE levels in mice after different experimental treatments.

[0043] Figure 12 The levels of cytokines in mice after different experimental treatments; Figure 12 In this context, A represents the level of the mouse cytokine IL-13 after different experimental treatments; Figure 12 In this context, B represents the level of the mouse cytokine IL-4 after different experimental treatments; Figure 12 In this context, C represents the level of the mouse cytokine IL-5 after different experimental treatments; Figure 12 In this context, D represents the level of the mouse cytokine IFN-γ after different experimental treatments. Detailed Implementation

[0044] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention. However, the content of the present invention is not limited to the scope described in the embodiments. Any changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0045] The adrenaline used in the examples is pharmaceutical-grade adrenaline free alkaloid (content ≥98.5%), pursinolide and isopursinolide are standards extracted and purified from bamboo leaves or rush pith (HPLC purity ≥95%), puerarin is a standard extracted and purified from kudzu root (HPLC purity ≥95%), vitexin is a standard extracted and purified from vitex (HPLC purity ≥95%), and isovitexin is a standard extracted and purified from hawthorn (HPLC purity ≥95%).

[0046] Example 1: Preparation of the adrenaline-isoscarpine supramolecular complex 0.461 g (2.10 mmol) of adrenaline and 0.942 g (2.10 mmol) of isopropargyl glycoside were added to a 600 mL polytetrafluoroethylene resonant cavity, along with 15 mm diameter zirconia ball grinding media, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonant mixer, the resonant frequency was set to 30 Hz, and the mixture was treated for 30 min. After treatment, the complex powder was collected and passed through a 100-mesh sieve to obtain 1.322 g of the adrenaline-isopropargyl glycoside supramolecular complex, with a yield of 94.2%.

[0047] Example 2: Preparation of the adrenaline-honeysuckle supramolecular complex 0.261 g (1.19 mmol) of adrenaline and 0.534 g (1.19 mmol) of arbutin were added to a 600 mL polytetrafluoroethylene resonant cavity, along with 15 mm diameter zirconia balls as the grinding medium, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonant mixer, and the resonant frequency was set to 30 Hz for 30 min. After treatment, the complex powder was collected and passed through a 100-mesh sieve to obtain 0.758 g of the adrenaline-arbutin supramolecular complex, with a yield of 95.3%.

[0048] Example 3: Preparation of the adrenaline-puerarin supramolecular complex 0.514 g (2.34 mmol) of adrenaline and 0.974 g (2.34 mmol) of puerarin were added to a 600 mL polytetrafluoroethylene (PTFE) resonant cavity, along with 15 mm diameter zirconia balls as the grinding medium, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonant mixer, and the resonant frequency was set to 30 Hz for 30 min. After treatment, the complex powder was collected and passed through a 100-mesh sieve to obtain 1.460 g of the adrenaline-puerarin supramolecular complex, with a yield of 98.1%.

[0049] Example 4: Preparation of the adrenaline-vitexin supramolecular complex 0.600 g (2.73 mmol) of epinephrine hydrochloride and 1.180 g (2.73 mmol) of vitexin were added to a 600 mL polytetrafluoroethylene resonant cavity, along with 15 mm diameter zirconia balls as the grinding medium, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonant mixer, and the resonant frequency was set to 30 Hz for 30 min. After treatment, the composite powder was collected and passed through a 100-mesh sieve to obtain 1.682 g of the epinephrine-vitexin supramolecular complex, with a yield of 94.5%.

[0050] Example 5: Preparation of the adrenaline-isoviticin supramolecular complex 0.340 g (1.55 mmol) of adrenaline and 0.670 g (1.55 mmol) of isovitelline were added to a 600 mL polytetrafluoroethylene resonant cavity, along with 15 mm diameter zirconia balls as the grinding medium, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonant mixer, and the resonant frequency was set to 30 Hz for 30 min. After treatment, the composite powder was collected and passed through a 100-mesh sieve to obtain 0.951 g of the adrenaline-isovitelline supramolecular complex, with a yield of 94.2%.

[0051] Example 6: Screening of flavonoid C-glycosides required for complex formation Following the methods in Examples 1-5, complexes of adrenaline and their corresponding C-glycosides were prepared, ensuring a molar ratio of 1:1. The powders of each complex were collected, and the saturated solubility of adrenaline in aqueous solution and its retention rate in aqueous solution within one month were determined. Each group was measured in triplicate, and the results were expressed as mean ± 95% confidence interval. The optimal C-glycoside for the complex was determined to be isoxalool, which had an adrenaline solubility of 1670 ± 22 mg / L and a retention rate of over 99.7% within one month.

[0052] Table 1 Group Adrenaline-flavonoid C-glycoside complex Solubility (mg / L) Retention rate within 1 month (%) Example 1 Adrenaline-Isopropanol 1670±22 99.70±0.21 Example 2 Adrenaline-Hydroxypropionate 1352±48 99.50±0.32 Example 3 Adrenaline-Puerariae 751±31 99.21±0.25 Example 4 Adrenaline-Vitexin 1171±44 99.31±0.12 Example 5 Adrenaline-Isovitilione 1525±27 98.24±0.16 adrenaline free base / 183±15 95.22±0.92 Example 7: Preparation of complexes with different molar ratios Following the method in Example 1, complexes of adrenaline and isohypoglycine in molar ratios of 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 were prepared, with other conditions remaining constant. The powders of each complex were collected for subsequent characterization and stability evaluation.

[0053] like Figure 1 As shown, complexes of adrenaline and isoharmone were prepared in different proportions. The solubility of adrenaline was affected by its molar ratio with isoharmone. It was found that the solubility was optimal when the molar ratio was 1:1. Since the solubility of both isoharmone and adrenaline is limited, the solubility decreased significantly when the molar ratio decreased or increased.

[0054] Example 8: Screening of acoustic resonance frequencies Following the method in Example 1, acoustic resonance frequencies were set to 10, 20, 25, 30, 35, and 40 Hz, while other conditions remained constant. After treatment, the saturated solubility and retention rate of adrenaline were measured, and the results are as follows: Figure 2 As shown in the figure. Experimental results show that the solubility and retention rate of the complex are optimal at a frequency of 30 Hz. At lower frequencies, the solubility decreases due to uneven mixing, while at higher frequencies, although the reaction is already uniform and the solubility increases, there is a large amount of frictional heat, which leads to the degradation of adrenaline, resulting in a further decrease in solubility and a severe reduction in retention rate. Therefore, 30 Hz is preferred as the optimal frequency.

[0055] Example 9: Screening of Processing Time Following the method in Example 1, processing times were set to 5, 10, 15, 30, 60, and 120 min, respectively, while keeping other conditions constant. The saturated solubility and retention rate of adrenaline were measured. The results are as follows: Figure 3The results show that the treatment time affects the solubility of the complex. Extending the treatment time significantly increases the solubility of the complex. After 30 min of treatment, the solubility of the complex basically stabilizes. Further extending the treatment time does not significantly improve it. Moreover, as the treatment time is extended, adrenaline begins to degrade, which is reflected in the reduced retention rate. In order to balance solubility and stability, 30 min is preferred as the optimal treatment time.

[0056] Example 10: Preparation of acoustic resonance mixed single-treatment adrenaline, acoustic resonance mixed single-treatment isopropanol, and physical mixture of adrenaline and isopropanol. 0.461 g (2.10 mmol) of epinephrine hydrochloride and 15 mm diameter zirconia ball grinding media were added to a 600 mL polytetrafluoroethylene resonant cavity, with a media-to-material mass ratio of 10:1. The resonant cavity was placed in an acoustic resonance mixer, the resonant frequency was set to 30 Hz, and the treatment was carried out for 30 min. After treatment, the complex powder was collected and passed through a 100-mesh sieve to obtain the control acoustic resonance mixed RAM with single-treated epinephrine, which was used for subsequent experiments.

[0057] Isophoramin was added to a 600 mL polytetrafluoroethylene (PTFE) resonant cavity. 0.942 g (2.10 mmol) of isophoramin was used as the grinding medium, with 15 mm diameter zirconia balls, and the mass ratio of medium to material was set to 10:1. The resonant cavity was placed in an acoustic resonant mixer, and the resonant frequency was set to 30 Hz for 30 min. After treatment, the composite powder was collected and passed through a 100-mesh sieve to obtain RAM-treated isophoramin, which was used for subsequent experiments.

[0058] The two were mixed in an equimolar ratio as a physical mixture control for subsequent experiments.

[0059] Example 11, XRPD characterization The composite prepared in Example 1 was characterized using X-ray powder diffraction. The test conditions were: Cu Kα radiation, tube voltage 40 kV, tube current 40 mA, scanning range 2θ = 5°–880°, and scanning speed 4° / min. The results are as follows: Figure 4As shown, isopropionyl glycoside and adrenaline exhibit sharp characteristic diffraction peaks at 2θ values ​​of 13.36°, 14.10°, 16.15°, 18.01°, 21.09° and 13.10°, 22.03°, 25.68°, respectively, indicating that both raw materials are crystalline. The XRD pattern of the physical mixture is a simple superposition of the diffraction peaks of the two components; no new diffraction peaks appeared or existing characteristic peaks disappeared, indicating that the simple physical mixing did not change their respective crystal structures. Notably, after acoustic resonance mixing treatment, the characteristic crystalline diffraction peaks of both components in the XRD pattern of Example 1 completely disappeared, replaced by broadened dispersion at approximately 26° 2θ, exhibiting typical amorphous characteristics. These results indicate that acoustic resonance mixing treatment effectively disrupts the long-range ordered crystal structure of isopropionyl glycoside and adrenaline, promoting the transformation of the system into an amorphous complex, forming a co-amorphous system.

[0060] Example 12, FT-IR characterization The complex prepared in Example 1 was characterized using Fourier transform infrared spectroscopy. The test conditions were: KBr pellet method, scanning range 4000-400 cm⁻¹. -1 4 cm resolution -1 The result is as follows Figure 5 As shown, isoharonin was at 3329 cm⁻¹ -1 The peak at 1699 cm⁻¹ shows a broad OH stretching vibration of phenolic and glycosylated hydroxyl groups. -1 The peak at 1507–1588 cm⁻¹ corresponds to the stretching vibration of the carbonyl group (C=O) at the 4-position of flavonoids. -1 The multiple peaks within this range are attributed to the C=C skeletal vibration of the aromatic rings, 1038–1138 cm⁻¹. -1 The sharp absorption peak in this region is a typical fingerprint characteristic of the COC and C-OH stretching vibrations in the C-glycoside structure; adrenaline at 3139 cm⁻¹ -1 The peaks at 1546–1587 cm⁻¹ represent the OH / NH stretching vibrations of phenolic hydroxyl groups, alcoholic hydroxyl groups, and amino groups. - ¹Corresponds to the C=C skeletal vibration of the benzene ring, 1272 cm⁻¹ -1 and 1029~1088 cm -1 These peaks are attributed to the stretching vibrations of phenol CO and alcohol CO, respectively. The infrared spectrum of the physical mixture shows a simple superposition of the characteristic peaks of the two components, with the peak positions being essentially consistent with those of the individual components, indicating that no significant intermolecular interactions have occurred. In stark contrast, the spectrum of the complex (Example 1) shows a significant difference: in the 3600–3000 cm⁻¹ range... -1 In this region, the OH stretching vibration peak is at 3136 cm⁻¹. - ¹Displaced to 3155 cm -1 The wavenumber shift is approximately 19 cm.-1 Meanwhile, the peak intensity decreased from 77.4 to 46.4, indicating that the hydroxyl group participated in the formation of intermolecular hydrogen bonds; at 1500~1600 cm⁻¹ -1 In the aromatic ring skeletal vibration region, the peak intensity decreased by approximately 18%, suggesting a possible π-π stacking interaction between the flavonoid aromatic ring and the benzene ring; this was particularly pronounced in the fingerprint region (1500–400 cm⁻¹). -1 The change in length is 1200~1500 cm. -1 The number of resolvable absorption peaks decreased from 11 to 6 within the range of 900–1200 cm⁻¹. -1 The number of glycosyl characteristic peaks decreased from 12 to 9, while the number of peaks in the 400–900 cm⁻¹ range increased. -1 The number of low-wavenumber skeletal vibrational peaks decreased sharply from 25 to 12, and the overall spectrum showed a significant broadening trend. This indicates that during ball milling, mechanical forces drove the two molecules to close contact and oriented alignment, forming a supramolecular structure with a specific spatial configuration. Intermolecular interactions restricted the degrees of freedom of some vibrational modes. Furthermore, the glycosyl-COC vibrational region (1003 cm⁻¹) -1 The absorbance difference reached 44.4, further confirming that the glycosyl group participated in the construction of the supramolecular complex. These results indicate that the isosorbide-adrenaline supramolecular complex was successfully prepared using the acoustic resonance mixing technique. The two components formed a stable complex system through non-covalent interactions such as hydrogen bonding and π-π stacking, rather than a simple physical mixture.

[0061] Example 13, DSC characterization The composite prepared in Example 1 was characterized using differential scanning calorimetry (DSC). The test conditions were: nitrogen atmosphere, heating rate 10°C / min, temperature range 150–300°C. The results are as follows: Figure 6 As shown, both adrenaline and isohypogonin exhibit distinct melting endothermic peaks at 211°C and 235°C, respectively, consistent with reported melting points. The complex, however, shows a distinct endothermic peak at approximately 218°C with decreasing intensity, indicating a different melting endothermic pattern compared to adrenaline and isohypogonin. This suggests the formation of a new, potentially supramolecular structure, further confirming the formation of the supramolecular complex.

[0062] Example 14, Particle Size Measurement The particle size distribution of the composite prepared in Example 1 was determined using a laser particle size analyzer. The experimental results are as follows: Figure 7 10 mg of the complex powder was dispersed in 10 mL of deionized water and ultrasonically dispersed for 5 min before analysis. The results showed that the complex had a relatively uniform particle size distribution, with a main average particle size of 194.7 nm ± 12.7 nm. The relatively uniform particle size distribution and PDI = 0.040 indicate that the treatment achieved mutual solubilization, which is beneficial to improving the solubility and bioavailability of the drug.

[0063] Example 15, Accelerated Stability Test The complex prepared in Example 1, the physical mixture (a simple mixture of adrenaline and isosorbide dinitrate, molar ratio 1:1), and the pure adrenaline raw material were placed in an accelerated stability test chamber at 40°C ± 2°C and 75% ± 5% relative humidity. Samples were taken at 0, 1, 2, 3, and 6 months, and the adrenaline content was determined by HPLC. The results are as follows: Figure 8 As shown, the stability of the complex was significantly better than that of the physical mixture and the active pharmaceutical ingredient. After 6 months, the adrenaline content in the complex remained above 97.3 ± 1.24%, while the content of the active pharmaceutical ingredient decreased to below 75%.

[0064] Example 16: Animal Model Experiment of Anaphylactic Shock 0.62 g of the adrenaline-isoharonidine supramolecular complex prepared in Example 1 (containing 0.22 g of adrenaline) was dissolved in 100 mL of water for injection. 0.9 g of sodium chloride was added to adjust to isotonicity, and the pH was adjusted to 3.5-4.0 with 0.1 M hydrochloric acid. After filtration through a 0.22 μm microporous membrane, the solution was dispensed into 1 mL ampoules, each containing 1 mg of adrenaline (calculated as free base). After sealing, the solution was sterilized at 121°C for 15 min to obtain the adrenaline-isoharonidine complex injection solution.

[0065] The efficacy of the complex prepared in Example 1 was evaluated using a mouse anaphylactic shock model. BALB / c mice were randomly divided into five groups: normal control group, model control group, adrenaline group, isopropanol group, and complex group, with 10 mice in each group. Sensitization was achieved by intraperitoneal injection of ovalbumin, followed by intravenous injection of ovalbumin two weeks later to induce anaphylactic shock. Immediately after stimulation, the mice were administered the drug intraperitoneally (the adrenaline group received an equivalent dose of adrenaline, and the complex group received an equivalent dose of the complex). The survival rate of each group was observed. Results are as follows: Figure 9 As shown, the survival rate of mice in the model group was low, only about 12%, while the survival rate of mice treated with commercially available epinephrine hydrochloride alone reached about 60%, and the survival rate of mice in the isoharmone-only group was only 23%, indicating that isoharmone itself has no significant therapeutic effect on anaphylactic shock. The survival rate of the complex group reached over 80%, significantly higher than that of the epinephrine-only group, indicating that isoharmone synergistically enhances the therapeutic effect of epinephrine on shock in mice through a complex formulation.

[0066] Example 17: Experimental Treatment of Allergic Asthma Female C57BL / 6 mice (18-22 g) aged 6-8 weeks were randomly divided into 6 groups (n=8 per group): normal control group, OVA model group, dexamethasone positive control group (10 mg / kg), adrenaline monotherapy group (0.1 mg / kg), isopropionate monotherapy group (50 mg / kg), physical mixture group, and Example 1 group (adrenaline dosage 0.1 mg / kg). Ovalbumin (OVA) sensitization was performed. On days 0, 7, and 14, mice in the model group and each drug-treated group were intraperitoneally injected with the sensitization solution (20 μg OVA + 1 mg aluminum hydroxide adjuvant dissolved in 0.2 mL physiological saline); the normal control group was injected with an equal volume of physiological saline. From days 21 to 28, mice were placed in a nebulizer and challenged with 1% OVA solution via nebulization (30 min / time, once daily); the normal control group was nebulized with physiological saline. Each treatment group was administered the drug 30 minutes before each OVA nebulization challenge via intraperitoneal injection, while the normal control group and model group received an equal volume of solvent. Administration continued for 7 days.

[0067] Twenty-four hours after the last challenge, the following procedures were performed: Bronchoalveolar lavage fluid (BALF): Endotracheal intubation was performed, and the bronchoscopy was repeated three times with 0.5 mL of pre-cooled PBS. The recovered fluids were combined for cell counting and inflammatory factor detection. Serum: Blood was collected by enucleation, centrifuged at 3000 rpm for 10 min, and the supernatant was used for IgE and inflammatory factor detection. The right lung was flash-frozen in liquid nitrogen for oxidative stress markers and protein detection. BALF was centrifuged at 1500 rpm for 5 min, and the precipitated cells were resuspended and counted. Cell suspension smears were prepared, stained with Wright-Giemsa, and the total number of cells, eosinophils, neutrophils, lymphocytes, and macrophages were counted under a microscope. Serum total IgE and OVA-specific IgE levels were detected using ELISA. IL-4, IL-5, IL-13, IL-17, TNF-α, and IFN-γ levels in BALF and serum were detected using ELISA.

[0068] Implementation results description, such as Figures 10-12 Compared with the OVA model group, the Example 1 group showed a significant reduction in the total number of cells and eosinophils in BALF; serum total IgE levels decreased, and this effect was superior to that of the raw materials epinephrine, isopropanol, and the physical mixture. Simultaneously, the levels of Th2 cytokines (IL-4, IL-5, IL-13) and the levels of Th1 cytokines (IFN-γ) in the BALF of the Example 1 group were decreased, and the Th1 / Th2 balance was restored. The results indicate that the epinephrine-isopropanol complex can significantly improve OVA-induced allergic asthma in mice by synergistically exerting bronchodilatory and anti-inflammatory effects, and its effect is superior to that of single epinephrine and the physical mixture.

[0069] In summary, this invention successfully prepared an adrenaline-flavonoid C-glycoside supramolecular complex using acoustic resonance mixing technology. This complex exhibits excellent stability, good solubility-release properties, and synergistic anti-inflammatory effects, effectively addressing the stability issues of adrenaline and providing a more reliable drug option for the rescue of critical illnesses such as anaphylactic shock and asthma. The preparation process of this invention is simple, environmentally friendly, and has promising prospects for industrial application.

Claims

1. A method for preparing an adrenaline-flavonoid C-glycoside supramolecular complex using acoustic resonance mixing technology, characterized in that, include: Adrenaline and flavonoid C-glycosides were mixed evenly at a molar ratio of 1:0.5 to 1:3 and placed in the resonance cavity of an acoustic resonance mixer. Grinding media was added, and the mixture was subjected to resonance mixing at a resonance frequency of 10 to 40 Hz for 5 to 120 min. After the treatment, the complex powder was collected to obtain the adrenaline-flavonoid C-glycoside supramolecular complex.

2. The preparation method according to claim 1, characterized in that, The adrenaline is pharmaceutical-grade free adrenaline base; The flavonoid C-glycosides are selected from one of the following: purslane, isopurslane, puerarin, vitexin, and isovitexin.

3. The preparation method according to claim 1, characterized in that, The molar ratio of adrenaline to flavonoid C-glycoside is 1:

1.

4. The preparation method according to claim 1, characterized in that, The resonant cavity of the acoustic resonance mixer is made of polytetrafluoroethylene or stainless steel, and its volume is 100~1000 mL.

5. The preparation method according to claim 1, characterized in that, The grinding media are zirconia balls, agate balls, or ceramic balls with a diameter of 10-25 mm.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the grinding media to the material is 1:1 to 20:

1.

7. The preparation method according to claim 1, characterized in that, The resonant frequency is 30 Hz.

8. The preparation method according to claim 1, characterized in that, The resonant mixing time is 30 min.

9. The use of the adrenaline-flavonoid C-glycoside supramolecular complex prepared according to claim 1 in the preparation of drugs for treating anaphylactic shock and / or allergic asthma.

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