Preparation method and application of effective part of hyssop officinalis
The effective components of *Hedyotis diffusa* were extracted by ethanol reflux and ethyl acetate extraction, which solved the shortcomings of *Hedyotis diffusa* in the treatment of allergic asthma and achieved significant anti-inflammatory and anti-asthmatic effects.
Patent Information
- Application Number
- CN202511378377.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2026-01-09
AI Technical Summary
In the existing technology, the application of *Hedyotis diffusa* in the treatment of allergic asthma has not been fully developed, and there is a lack of effective preparation methods to exert its anti-inflammatory, antitussive, and bronchodilator pharmacological effects.
The effective components of *Hedyotis diffusa* were extracted by ethanol reflux extraction and ethyl acetate extraction to prepare *Hedyotis diffusa* ethanol extract. The effective components of *Hedyotis diffusa* were then obtained by vacuum drying and vacuum concentration.
It significantly reduced lung injury and inflammatory response in mice with allergic asthma, downregulated the levels of related cytokines, and reduced the number of inflammatory cells, demonstrating a significant anti-asthmatic effect.
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Figure CN121287775A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing the effective fraction of *Hedyotis diffusa* and its application. Background Technology
[0002] Asthma is a common chronic inflammatory disease of the airways, characterized by airway hyperresponsiveness and airway remodeling. Hyssopus cuspidatus Boriss., a plant in the Lamiaceae family, is a dried aerial part of the herb. It is hot and dry in nature, aromatic, and bitter in taste. It has antitussive, expectorant, and bronchodilator effects, and promotes the body's natural harmonization. It has been widely used in folk medicine and clinical practice. The whole herb of Hyssopus cuspidatus contains volatile oils with antispasmodic alkaloid-like effects, which can relieve bronchospasm and also exhibit antitussive and expectorant functions. Modern pharmacology has also confirmed that Hyssopus cuspidatus has anti-inflammatory, antitussive, expectorant, and bronchodilator effects. This study observed the effects of the effective parts of Hyssopus cuspidatus on lung injury in mice, allergic asthma-related cytokines, and inflammatory cells in bronchoalveolar lavage fluid (BALF). The results reveal that the effective parts of Hyssopus cuspidatus have a good interventional effect on allergic asthma. Summary of the Invention
[0003] The purpose of this invention is to provide a method for preparing and applying the effective components of *Hedyotis diffusa*. The method involves cutting *Hedyotis diffusa* into segments, extracting it by reflux with ethanol, filtering, combining the filtrates, concentrating under reduced pressure, and drying under vacuum to obtain an ethanol extract of *Hedyotis diffusa*. The extract is then dissolved in ethanol, extracted with ethyl acetate, and the upper layer is concentrated under reduced pressure and dried under vacuum to obtain the effective components of *Hedyotis diffusa*.
[0004] The method for preparing the effective part of *Hedyotis diffusa* according to the present invention comprises the following steps:
[0005] a. Cut the herb *Hedyotis diffusa* into 1-inch segments, and extract it by reflux with 15-20 times the amount of 50% ethanol for 2-3 times, each time for 1-3 hours. Filter, combine the filtrates, concentrate under reduced pressure, and dry under vacuum at 50℃ and 0.08 MPa to obtain a 50% ethanol extract of *Hedyotis diffusa*.
[0006] b. Dissolve the extract obtained in step a in 30% ethanol, then extract with ethyl acetate 2-3 times, 50-150 ml each time. Take the upper layer and concentrate under reduced pressure, dry at 50℃ and 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
[0007] The application of the effective fraction of *Hedyotis diffusa* obtained by the method in the preparation of anti-allergic asthma drugs. Attached Figure Description
[0008] Figure 1 This is an HE staining image of lung tissue from the present invention;
[0009] Figure 2 The present invention SXCH can downregulate the increase of serum IgE in mice with OVA-induced allergic asthma, and at the same time reverse the increase of inflammation and immune-related cytokines IFN-γ, IL-1β, IL-5, IL-6 and TNF-α in BALF of mice with OVA-induced allergic asthma.
[0010] Figure 3 The SXCH of this invention can significantly reduce eosinophils, neutrophils, and basophils in the BALF of OVA-induced allergic asthma mice;
[0011] Figure 4 This is the SXCH feature map of the present invention;
[0012] Figure 5 This is the HPLC chromatogram of the mixed reference standard of the present invention. Detailed Implementation
[0013] Example 1
[0014] a. Take 150g of *Hedyotis diffusa* herb, cut it into 1-inch sections, and extract it twice with 15 times the amount of 50% ethanol, each time for 1 hour. Filter, combine the filtrates, concentrate under reduced pressure, dry under vacuum at 50℃ and 0.08MP to obtain 50% ethanol extract of *Hedyotis diffusa*.
[0015] b. Dissolve the extract obtained in step a in 30% ethanol, then extract twice with ethyl acetate, 50 ml each time. Take the upper layer and concentrate under reduced pressure, dry at 50℃ and 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
[0016] Example 2
[0017] a. Take 150g of the herb *Hedyotis diffusa*, cut it into 1-inch sections, and extract it three times with 18 times the amount of 50% ethanol, each time for 2 hours. Filter, combine the filtrates, concentrate under reduced pressure, dry at 50℃ and 0.08MPa vacuum to obtain the 50% ethanol extract of *Hedyotis diffusa*.
[0018] b. Dissolve the extract obtained in step a in 30% ethanol, then extract with ethyl acetate three times, 100 ml each time. Take the upper layer and concentrate under reduced pressure, dry at 50°C and 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
[0019] Example 3
[0020] a. Take 150g of the herb *Hedyotis diffusa*, cut it into 1-inch sections, and extract it three times with 20 times the amount of 50% ethanol, each time for 3 hours. Filter, combine the filtrates, concentrate under reduced pressure, dry at 50℃ and 0.08MPa vacuum to obtain the 50% ethanol extract of *Hedyotis diffusa*.
[0021] b. Dissolve the extract obtained in step a in 30% ethanol, then extract with ethyl acetate three times, 150 ml each time. Take the upper layer and concentrate under reduced pressure, dry at 50°C and 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
[0022] Example 4
[0023] a. Take 150g of the herb *Hedyotis diffusa*, cut it into 1-inch sections, and extract it twice with 17 times the amount of 50% ethanol, each time for 3 hours. Filter, combine the filtrates, concentrate under reduced pressure, dry under vacuum at 50℃ and 0.08MP to obtain the 50% ethanol extract of *Hedyotis diffusa*.
[0024] b. Dissolve the extract obtained in step a in 30% ethanol, then extract twice with ethyl acetate, 80 ml each time. Take the upper layer and concentrate under reduced pressure, dry at 50°C and 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
[0025] Example 5
[0026] Evaluation of the efficacy of the effective component of the herb *Hedyotis diffusa* in treating allergic asthma according to this invention:
[0027] 1.1 Materials:
[0028] 1.1.1 Laboratory animals:
[0029] BALB / c mice, weighing 18–20 g, female, were provided by the Animal Experiment Center of Xinjiang Medical University, totaling 92 mice. Ethics review number: XJIMM-20230704.
[0030] 1.1.2 Experimental drugs and reagents:
[0031] The positive control drug, dexamethasone, was prepared by crushing the tablets into powder and reconstituted with 0.5% CMC-Na solution to a gavage dose of 1.6 mg / kg / day. -1 Aluminum hydroxide gel diluted with physiological saline, ovalbumin (OVA), sterile physiological saline, ELISA kit, 4% paraformaldehyde, hematoxylin-eosin (HE) staining kit, xylene, anhydrous ethanol, 90% ethanol, 70% ethanol, reference standards chlorogenic acid, isochlorogenic acid, caffeic acid, geraniol, trifurciferol, isoquercitrin, rosmarinic acid, neochlorogenic acid, farnesin, and acetonitrile were all of chromatographic grade and phosphoric acid analytical grade. The effective components of *Hedyotis diffusa* were also used.
[0032] 1.1.3 Main Instruments:
[0033] BH-2 biological microscope, paraffin sectioning apparatus, centrifuge, incubator, shaker, Agilent 1260 high-performance liquid chromatograph (1260 Infinity Agilent Technologies); TE212-L electronic balance (Sartorius Scientific Instruments Co., Ltd.); PA225 D electronic balance (Sartorius Scientific Instruments, Germany); KQ-500VDV ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., Kunshan).
[0034] 1.2 Methods:
[0035] 1.2.1 Grouping and Dosing:
[0036] After 6 days of adaptive feeding, mice were randomly divided into four groups based on body weight: control group (Control), model group (Model), low-dose group of effective part of *Hydrocotyle sibirica* (L-dose), medium-dose group of effective part of *Hydrocotyle sibirica* (M-dose), high-dose group of effective part of *Hydrocotyle sibirica* (H-dose), and positive control group (Dex), with 12 mice in each group.
[0037] 1.2.2 Establishment of a mouse model of allergic asthma:
[0038] An OVA-induced allergic asthma mouse model was established. On days 8 and 15, mice in the model group were intraperitoneally injected with 0.2 mL of sensitizing solution (0.5 mg / mL). -1 Mice were sensitized twice with OVA (aluminum hydroxide gel diluted with physiological saline), and the normal control group mice were injected with an equal volume of physiological saline; from day 22 to 28, the model group mice were nebulized with a challenge solution (10 mg / mL). -1 OVA) was continuously challenged for 7 days, once a day for 30 minutes each time. The normal control group mice were challenged with physiological saline. During the period, the mice had free access to food and water.
[0039] 1.2.3 Behavioral assessment:
[0040] After stimulating mice with OVA nebulized stimulating solution, it was observed that the mice exhibited obvious symptoms such as wheezing, difficulty breathing, restlessness, frequent scratching of ears and nose, sneezing, and rapid breathing.
[0041] 1.2.4 Collection and pretreatment of various biological samples:
[0042] Biological samples were collected from each group 24 hours after the last excitation.
[0043] Serum: Blood was collected from mice after removing their eyeballs and placed in Eppendorf tubes. The tubes were left to stand at room temperature for 2 hours, then centrifuged at 3000 r / min for 15 min. The serum was then stored at -20℃.
[0044] Lung tissue: After blood collection, the mice were euthanized by cervical dislocation, lung tissue was collected, rinsed with physiological saline, and placed in 5 mL Eppendorf tubes (containing 4% paraformaldehyde solution) for lung tissue morphology analysis.
[0045] Bronchoalveolar lavage fluid (BALF): Mice were euthanized by cervical dislocation. The peritracheal tissue was immediately separated, and a transverse incision was made in the upper trachea (between the third and fourth tracheal rings). A syringe (1 mL) needle was inserted into the trachea (approximately 2 mm) and fixed. 1.5 mL of physiological saline was injected into the lungs, and the lavage was repeated three times. BALF was centrifuged at 2000 rpm for 10 min twice. The supernatant was aliquoted into 200 μL tubes and stored at -80°C for the determination of TNF-α, IL-6, and IFN-γ levels.
[0046] 1.3 Results:
[0047] 1.3.1 Lung tissue morphological analysis:
[0048] Lung tissue was fixed with 4% paraformaldehyde for 48 hours with gentle shaking, then dehydrated, embedded, sectioned, stained with hematoxylin and eosin (HE), and examined for lung tissue damage under a light microscope. The results are as follows: Figure 1 ;
[0049] 1.3.2 Detection of BALF and serum cytokines:
[0050] The levels of IFN-γ, IL-1β, IL-5, IL-6, TNF-α in BALF and serum IgE were measured using the ELISA kit according to the instructions. The results are as follows: Figure 2 ;
[0051] 1.3.3 Detection of the number of immune cells in BALF:
[0052] The number of eosinophils, neutrophils, and basophils in mouse bronchoalveolar lavage fluid was determined using a cell counting method. The results are as follows: Figure 3 ;
[0053] HE staining analysis revealed that, compared to the control group, the model group showed extensive inflammatory cell infiltration of the bronchial and perivascular areas, inflammatory cell exudation in the bronchial lumen, bronchial epithelial cell degeneration, and mucosal thickening. Furthermore, the number of eosinophils, neutrophils, and basophils in the bronchoalveolar lavage fluid (BALF) was significantly increased, serum IgE levels were elevated, and the levels of inflammation- and immune-related cytokines IFN-γ, IL-1β, IL-5, IL-6, and TNF-α in BALF were also elevated. After administration of SXCH, the pathological condition of mice was effectively alleviated compared to the model group, lung injury showed varying degrees of recovery, serum IgE levels were downregulated, and the levels of related inflammatory factors decreased. These experimental results indicate that the effective components of *Hedyotis diffusa* have a significant anti-asthmatic effect.
[0054] Establishment of characteristic spectra of effective components of *Hedyotis diffusa* using HPLC technology:
[0055] 2.1 Chromatographic conditions:
[0056] COSMOSIL C 18 Chromatographic column (250×4.6mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution (0-15min, 10% A; 15-25min, 90% A→87% A; 25-38min, 87% A; 38-60min, 87% A→80% A; 60-70min, 80% A→70% A; 75-80min, 35% A→40% A; 80-90min, 40% A→50% A; 90-100min, 50% A; 100-100.1min, 50% A→10% A; 100.1-105min, 10% A); column temperature 30℃; flow rate 1.0mL / min; detection wavelength 330nm; injection volume 20μL.
[0057] 2.2 Preparation of mixed reference solution:
[0058] Accurately weigh appropriate amounts of the reference standards chlorogenic acid, isochlorogenic acid, caffeic acid, geraniol, trifurciferol, isoquercitrin, rosmarinic acid, neochlorogenic acid, and farnesin, and dissolve them in 50% methanol to prepare a solution of 0.1 mg / mL. -1 A mixed reference solution;
[0059] 2.3 Preparation of the test solution:
[0060] Accurately weigh 20 mg of the effective part of *Hedyotis diffusa* and place it in a 10 ml volumetric flask. Add 10 mL of 50% methanol solution, weigh, and treat with ultrasound at 300 W and 45 kHz for 10 min. Let it cool, weigh it again, and replenish the lost amount with 50% methanol aqueous solution. Shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.
[0061] 2.4 HPLC characteristic chromatograms of the effective fraction of *Hedyotis diffusa*:
[0062] Take the effective parts of *Hedyotis diffusa*, prepare the test solution according to the method in section 2.3, and perform the determination under the chromatographic conditions determined in section 2.1. Record the chromatogram of the sample, and the results are as follows: Figure 4 ;
[0063] Figure 4 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Caffeic acid; 4. Isoquercitrin; 9. Rosmarinic acid; 10. Isochlorogenic acid; 11. Syringaresinol; 13. Scutellarein;
[0064] Based on the above method, it was determined that the chromatogram of the test sample should show 13 characteristic peaks. A mixed reference solution was prepared according to the method in section 2.2, and the determination was performed under the chromatographic conditions determined in section 2.1. The chromatogram of the sample was recorded, and the results are as follows: Figure 5 ;
[0065] Figure 5 1. Neochlorogenic acid; 2. Chlorogenic acid; 3. Caffeic acid; 4. Isoquercitrin; 5. Rosmarinic acid; 6. Isochlorogenic acid; 7. Syringaresinol; 8. Scutellarein;
[0066] The characteristic peaks of SXCH were identified as follows: peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is caffeic acid, peak 4 is isoquercitrin, peak 9 is rosmarinic acid, peak 10 is isochlorogenic acid, peak 11 is geraniol, and peak 13 is trifleurone.
Claims
1. A method for preparing the effective parts of *Hedyotis diffusa*, characterized in that... Follow these steps: a. Cut the herb *Hedyotis diffusa* into 1-inch segments, and extract it by reflux with 15-20 times the amount of 50% ethanol for 2-3 times, each time for 1-3 hours. Filter, combine the filtrates, concentrate under reduced pressure, and dry under vacuum at 50℃ and 0.08MP to obtain a 50% ethanol extract of *Hedyotis diffusa*. b. Dissolve the extract obtained in step a in 30% ethanol, then extract with ethyl acetate 2-3 times, 50-150 ml each time. Take the upper layer and concentrate under reduced pressure at 50℃ and dry under 0.08 MPa vacuum to obtain the effective part of *Hedyotis diffusa*.
2. The use of the effective fraction of *Hedyotis diffusa* obtained by the method described in claim 1 in the preparation of anti-allergic asthma drugs.