Preparation method and application of alpine horseradish water extract for relieving cough and resisting inflammation

Abstract

The invention discloses a preparation method and application of an alpine horseradish water extract for relieving cough and resisting inflammation, and relates to the technical field of preparation of cough-relieving and anti-inflammatory medicines. The traditional Chinese medicine composition comprises the following components in parts by weight: 60-80 parts of alpine horseradish, 25-35 parts of perilla leaves, 18-28 parts of bulbus fritillariae cirrhosae, 12-22 parts of rhizoma anemarrhenae, 10-20 parts of white mulberry root-bark, 8-15 parts of lumbricus, 6-12 parts of cynanchum glaucescens and 5-10 parts of blackberry lily. According to the alpine horseradish water extract disclosed by the invention, alpine horseradish is taken as a monarch drug, and rich flavonoid components in the alpine horseradish can effectively inhibit release of inflammatory factors and relieve cough reflex; ministerial drugs such as perilla leaves and bulbus fritillariae cirrhosae are supplemented, volatile oil components of the perilla leaves can enhance the relaxation effect of airway smooth muscles, and alkaloid of the bulbus fritillariae cirrhosae can cooperatively relieve a cough and dilute sputum; the components such as rhizoma anemarrhenae and the root bark of white mulberry are used as assistants, saponin substances of the rhizoma anemarrhenae can enhance the anti-inflammatory activity, and flavonoid components of the root bark of white mulberry can relieve airway mucosa edema; the earthworm, the cynanchum glaucescens and the blackberry lily are used, plasmin of the earthworm can improve airway microcirculation, and saponin of the cynanchum glaucescens can enhance the cough relieving effect.

Description

Technical Field

[0001] This invention relates to the field of antitussive and anti-inflammatory drug preparation technology, specifically to a method for preparing and applying an aqueous extract of horseradish for antitussive and anti-inflammatory purposes. Background Technology

[0002] Cough and inflammation are common clinical symptoms, often triggered by respiratory infections, allergies, and environmental irritants, significantly impacting patients' quality of life. Currently, commonly used cough suppressants mainly include centrally acting antitussives (such as codeine) and peripherally acting antitussives (such as dextromethorphan), while anti-inflammatory drugs primarily consist of nonsteroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen) and corticosteroids. However, these medications have certain limitations in their use: long-term use of centrally acting antitussives can easily lead to dependence and tolerance, while peripherally acting antitussives are often less effective; NSAIDs may cause gastrointestinal adverse reactions, and corticosteroids have side effects such as immunosuppression and endocrine disorders.

[0003] Traditional Chinese medicine has a long history of application in the fields of cough relief and anti-inflammation. Its multi-component and multi-target effects can effectively alleviate symptoms with minimal side effects. Horseradish, a traditional Tibetan medicine, has the effects of clearing heat and detoxifying, relieving cough and asthma. Modern pharmacological studies have shown that it contains active ingredients such as flavonoids and alkaloids, possessing certain potential for cough relief and anti-inflammation. However, current applications of horseradish are mostly focused on the extraction of single components or simple formulations, lacking scientifically sound compound design and efficient extraction and purification processes. This results in low utilization of its active ingredients and insufficient efficacy, failing to meet clinical needs.

[0004] Furthermore, existing methods for preparing traditional Chinese medicine extracts suffer from problems such as long extraction times, significant loss of active ingredients, and poor purification effects, which limit the quality improvement and clinical application of traditional Chinese medicine preparations. Therefore, developing a cough-relieving and anti-inflammatory aqueous extract with alpine horseradish as the main ingredient, combined with other effective traditional Chinese medicine components and advanced extraction and purification technologies, is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying an aqueous extract of horseradish for cough relief and anti-inflammation, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a water extract of horseradish for cough relief and anti-inflammation, characterized in that, by weight, it comprises the following components:

[0007] 60-80 portions of horseradish, 25-35 portions of perilla leaves, 18-28 portions of fritillaria cirrhosa, 12-22 portions of anemarrhena asphodeloides, 10-20 portions of mulberry bark, 8-15 portions of earthworm, 6-12 portions of cynanchum paniculatum, and 5-10 portions of belamcanda chinensis.

[0008] Preferably, the product comprises the following components in parts by weight:

[0009] 70 portions of horseradish, 30 portions of perilla leaves, 23 portions of fritillaria cirrhosa, 17 portions of anemarrhena asphodeloides, 15 portions of mulberry bark, 11 portions of earthworm, 9 portions of cynanchum paniculatum, and 7 portions of belamcanda chinensis.

[0010] A method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation includes the following steps:

[0011] Horseradish, perilla leaves and fritillaria cirrhosa are crushed and filtered separately, then mixed to obtain material A. Anemarrhena asphodeloides, mulberry bark and earthworm are crushed and filtered separately, then mixed to obtain material B. Cynanchum paniculatum and Belamcanda chinensis are crushed and filtered separately, then mixed to obtain material C.

[0012] After cleaning and removing impurities, materials A, B, and C are soaked in a 0.1%-0.3% sodium bicarbonate aqueous solution for 20-40 minutes, and then dried in a vacuum drying oven at 65-75℃ with a vacuum degree of 0.08-0.06 MPa.

[0013] Add the pretreated material A to a dynamic extraction tank, add 10-15 times its weight of purified water, set the extraction temperature to 90-95℃, the stirring speed to 60-80 r / min, and extract for 3-4 hours. After filtration, obtain filtrate A. Add 8-10 times its weight of purified water to the filter residue and extract for another 1.5-2 hours. After filtration, obtain filtrate A. / Combine filtrate A and filtrate A / Extract A was obtained;

[0014] Add 70%-70% ethanol aqueous solution to the pretreated material B, with a weight ratio of material B to ethanol aqueous solution of 1:8-12. Then add cellulase with an enzyme activity of 5000 U / g and pectinase with an enzyme activity of 3000 U / g, with a mass ratio of cellulase to pectinase of 1:1. The amount of enzyme added is 0.5%-1% of the weight of material B. After enzymatic hydrolysis at 45-55℃ for 2-3 hours, place it in an ultrasonic extractor, set the power to 400-500W and the frequency to 35±5KHz, and extract for 1-1.5 hours. After filtration, extract B is obtained.

[0015] Add the pretreated material C to a microwave extractor, add 6-8 times the weight of purified water, set the power to 600-700W, the temperature to 75-85℃, extract for 25-40 minutes, and filter to obtain extract C.

[0016] Mix extract A, extract B and extract C, concentrate under reduced pressure at 50-60℃ to a relative density of 1.15-1.25 to obtain a concentrate, add 2-3 times its volume of anhydrous ethanol to the concentrate, stir and let stand for 12-18 hours, centrifuge and take the supernatant, recover the ethanol under reduced pressure until there is no alcohol odor, to obtain the crude extract.

[0017] The crude extract was dissolved in purified water and loaded onto an HPD-100 macroporous resin column at a flow rate of 1-1.5 BV / h. After adsorption saturation, the column was eluted with 3-4 BV of purified water at a flow rate of 2-2.5 BV / h, followed by elution with 60%-70% ethanol aqueous solution at a flow rate of 1.5-2 BV / h for 4-5 BV. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain the alpine horseradish extract used for antitussive and anti-inflammatory purposes.

[0018] Preferably, material A passes through a 100-120 mesh sieve, material B passes through an 80-100 mesh sieve, and material C passes through a 60-80 mesh sieve.

[0019] Preferably, the mass concentration of the ethanol aqueous solution is 75%, and the weight ratio of material B to the ethanol aqueous solution is 1:10.

[0020] Preferably, the extraction parameters of the microwave extractor are 650W power, 80℃ temperature, and 30min extraction time.

[0021] Preferably, the freeze-drying time is 24-36 hours.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention discloses the preparation method and application of an aqueous extract of *Hordeum vulgare* (mountain horseradish) for cough suppression and anti-inflammation. The extract uses *Hordeum vulgare* as the principal ingredient, whose rich flavonoids effectively inhibit the release of inflammatory factors and alleviate the cough reflex. It is supplemented with herbs such as perilla leaf and fritillaria cirrhosa. The volatile oil components of perilla leaf enhance the relaxation of airway smooth muscle, while the alkaloids of fritillaria cirrhosa synergistically suppress cough and thin sputum. It is further enhanced with ingredients such as anemarrhena asphodeloides and mulberry bark. The saponins of anemarrhena asphodeloides enhance anti-inflammatory activity, and the flavonoids of mulberry bark reduce airway mucosal edema. Finally, it includes earthworm, cynanchum paniculatum, and belamcanda chinensis. The fibrinolytic enzymes of earthworm improve airway microcirculation, the saponins of cynanchum paniculatum enhance the antitussive effect, and the isoflavones of belamcanda chinensis inhibit airway inflammatory responses. This combination of herbs forms a multi-target mechanism of action, significantly improving the antitussive and anti-inflammatory effects and solving the problem of insufficient efficacy of single-component drugs.

[0024] Simultaneously, a multi-stage extraction technique was employed, involving graded pulverization, targeted pretreatment (soaking in sodium bicarbonate aqueous solution to remove impurities), and stepwise extraction. Material A underwent dynamic water decoction extraction to fully retain water-soluble active ingredients. Material B combined enzymatic hydrolysis and ultrasonic extraction, utilizing cellulase and pectinase to disrupt cell wall structures, along with the cavitation effect of ultrasound, to improve the dissolution rate of lipid-soluble components. Material C was extracted using microwaves, leveraging the thermal and non-thermal effects of microwaves to rapidly extract small-molecule active substances. Subsequent purification via macroporous resin adsorption and freeze-drying effectively removed impurities while retaining heat-sensitive components, resulting in extracts with high purity and good stability, overcoming the shortcomings of traditional extraction methods such as low efficiency and significant component loss. Detailed Implementation

[0025] This invention provides a technical solution:

[0026] Example 1:

[0027] A water extract of horseradish for cough relief and anti-inflammation, by weight, comprises: 70 parts horseradish, 30 parts perilla leaf, 23 parts fritillaria cirrhosa, 17 parts anemarrhena asphodeloides, 15 parts mulberry bark, 11 parts earthworm, 9 parts cynanchum paniculatum, and 7 parts belamcanda chinensis.

[0028] Its preparation method includes the following steps:

[0029] Horseradish, perilla leaves and fritillaria bulb are crushed separately, passed through a 100-120 mesh sieve, and then mixed to obtain material A;

[0030] Anemarrhena asphodeloides, mulberry bark, and earthworm are crushed separately, passed through an 80-100 mesh sieve, and then mixed to obtain material B;

[0031] The white powder and the belamcanda chinensis were crushed separately, passed through a 60-80 mesh sieve, and then mixed to obtain material C.

[0032] Materials A, B, and C were washed separately to remove impurities. They were then soaked in a 0.2% sodium bicarbonate aqueous solution for 30 minutes and then dried in a vacuum drying oven at 70°C with a vacuum degree of 0.07 MPa.

[0033] Pretreated material A was added to a dynamic extraction tank, and purified water (12 times its weight) was added. The extraction temperature was set to 92℃, the stirring speed to 70 r / min, and the extraction was carried out for 3.5 h. Filtrate A was obtained by filtration. Purified water (9 times its weight) was added to the filter residue, and the extraction was carried out again for 1.8 h using the above parameters. Filtrate A was obtained by filtration. / Combine filtrate A and filtrate A / Extract A was obtained;

[0034] Add 75% ethanol aqueous solution to pretreated material B, with a weight ratio of material B to ethanol aqueous solution of 1:10. Then add cellulase and pectinase, with a mass ratio of cellulase to pectinase of 1:1 and an enzyme addition amount of 0.7% of the weight of material B. After enzymatic hydrolysis at 50℃ for 2.5h, place it in an ultrasonic extractor, set the power to 450W and the frequency to 35kHz, and extract for 1.2h. Filter to obtain extract B.

[0035] The pretreated material C was added to a microwave extractor, along with 7 times the weight of purified water. The power was set to 650W and the temperature to 80℃. Extraction was carried out for 30 minutes, and the extract was filtered to obtain extract C.

[0036] Extracts A, B, and C were mixed and concentrated under reduced pressure at 55°C to a relative density of 1.20 to obtain the concentration. Anhydrous ethanol, 2.5 times its volume, was added to the concentrator, stirred evenly, and allowed to stand for 15 hours. After centrifugation, the supernatant was collected, and the ethanol was recovered under reduced pressure until no alcohol odor was detected to obtain the crude extract.

[0037] The crude extract was dissolved in purified water and loaded onto an HPD-100 macroporous resin column at a flow rate of 1.2 BV / h. After adsorption saturation, the column was eluted with 3.5 BV of purified water at a flow rate of 2.2 BV / h, followed by elution with 65% ethanol aqueous solution at a flow rate of 1.8 BV / h for a total of 4.5 BV. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain an aqueous extract of horseradish used for cough relief and anti-inflammation.

[0038] Example 2:

[0039] A water extract of horseradish for cough relief and anti-inflammation, by weight, comprises: 60 parts horseradish, 25 parts perilla leaf, 18 parts fritillaria cirrhosa, 12 parts anemarrhena asphodeloides, 10 parts mulberry bark, 8 parts earthworm, 6 parts cynanchum paniculatum, and 5 parts belamcanda chinensis.

[0040] The preparation method is basically the same as that in Example 1, except that the extraction time of material A is 3 hours and the extraction time is 1.5 hours.

[0041] Example 3:

[0042] A water extract of horseradish for cough relief and anti-inflammation, by weight, comprises: 80 parts horseradish, 35 parts perilla leaf, 28 parts fritillaria cirrhosa, 22 parts anemarrhena asphodeloides, 20 parts mulberry bark, 15 parts earthworm, 12 parts cynanchum paniculatum, and 10 parts belamcanda chinensis.

[0043] The preparation method is basically the same as that in Example 1, except that the enzymatic hydrolysis temperature is 45℃ and the enzymatic hydrolysis time is 3h.

[0044] Example 4:

[0045] A water extract of horseradish for cough relief and anti-inflammation, using the components of Example 1.

[0046] The preparation method is basically the same as that in Example 1, except that AB-8 type macroporous resin column is used.

[0047] Control group 1:

[0048] An extract was obtained by using a single type of alpine horseradish and extracting it according to the preparation method in Example 1.

[0049] Control group 2:

[0050] Using the components from Example 1, the traditional water decoction method was employed for extraction. After mixing all components, 10 times their weight of water were added, and the mixture was boiled and kept at a gentle boil for 2 hours. The mixture was then filtered, and the filtrate was concentrated under reduced pressure and dried to obtain the extract.

[0051] Cough-suppressing effect experiment:

[0052] One hundred and twenty Kunming mice (half male and half female, weighing 20±2g) were randomly divided into six groups of twenty mice each: Example 1 group, Example 2 group, Example 3 group, Example 4 group, Control Group 1, and Control Group 2. A blank control group (administered an equal volume of physiological saline) was also included. Mice in each group were administered the drug by gavage for seven consecutive days, once daily, at a dose of 20mg / kg. One hour after the last administration, the mice were placed in a glass bell jar, and coughing was induced by stimulating them with an ammonia-soaked cotton ball (0.2mL of 25% ammonia). The cough latency (time from the insertion of the cotton ball to the first cough) and the number of coughs within 2 minutes were recorded. The results are shown in Table 1 below.

[0053] Table 1 Comparison of cough-suppressing effects in different groups of mice (x±s)

[0054]

[0055]

[0056] As shown in Table 1, the cough latency period of each example group was significantly longer than that of the blank control group, and the number of coughs within 2 minutes was significantly less than that of the blank control group (P<0.01). The cough latency period of Example 1 group was the longest, the number of coughs was the fewest, and the cough-suppressing effect was the best. The cough-suppressing effects of Example 2 group, Example 3 group, and Example 4 group were slightly inferior to those of Example 1 group, but superior to those of Control Group 1 and Control Group 2 group. The cough-suppressing effects of Control Group 1 and Control Group 2 group were poor, indicating that the formula and preparation method of the present invention can significantly improve the cough-suppressing effect.

[0057] Anti-inflammatory effect experiment:

[0058] One hundred and twenty Kunming mice (half male and half female, weighing 20±2g) were randomly divided into six groups of twenty mice each, with grouping as in the cough-suppressing effect experiment. Mice in each group were administered the drug by gavage for seven consecutive days, once daily, at a dose of 20mg / kg. One hour after the last administration, 0.05mL of xylene was applied to both the anterior and posterior surfaces of the right ear of each mouse, with the left ear serving as a control. Four hours later, the mice were sacrificed, and ear flaps were punched from the same location in both ears using an 8mm diameter punch. The flaps were weighed, and the degree of ear swelling was calculated (right ear flap weight - left ear flap weight). The results are shown in Table 2 below.

[0059] Table 2 Comparison of anti-inflammatory effects in mice of different groups (x±s)

[0060] Group Ear swelling degree (mg) Blank control group 32.6±3.5 Example 1 Group 10.2±1.8 Example 2 group 13.5±2.1 Example 3 Group 12.1±1.9 Example 4 group 15.3±2.3 Control group 1 23.8±2.8 control group 2 20.5±2.5

[0061] As shown in Table 2, the ear swelling in each of the example groups was significantly lower than that in the blank control group (P<0.01); the ear swelling in the example 1 group was the smallest, and the anti-inflammatory effect was the best; the anti-inflammatory effects of the example 2, example 3, and example 4 groups were slightly inferior to those of the example 1 group, but superior to those of the control group 1 and control group 2; the anti-inflammatory effects of the control group 1 and control group 2 were poor, indicating that the formulation and preparation method of the present invention can significantly improve the anti-inflammatory effect.

[0062] The above experiments show that the alpine horseradish water extract of the present invention, used for cough relief and anti-inflammation, has significant cough relief and anti-inflammation effects. Among them, Example 1 has the best effect. Its formula is scientific and reasonable, its preparation process is advanced, and the components work synergistically to effectively exert cough relief and anti-inflammation effects. It is superior to extracts prepared by single components and traditional extraction methods and has good clinical application prospects.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended embodiments and their equivalents.

Claims

1. A water extract of horseradish for cough relief and anti-inflammation, characterized in that... It comprises the following components in parts by weight: 60-80 portions of horseradish, 25-35 portions of perilla leaves, 18-28 portions of fritillaria cirrhosa, 12-22 portions of anemarrhena asphodeloides, 10-20 portions of mulberry bark, 8-15 portions of earthworm, 6-12 portions of cynanchum paniculatum, and 5-10 portions of belamcanda chinensis.

2. The water extract of horseradish for cough relief and anti-inflammation according to claim 1, characterized in that, Based on parts by weight, it comprises the following components: 70 portions of horseradish, 30 portions of perilla leaves, 23 portions of fritillaria cirrhosa, 17 portions of anemarrhena asphodeloides, 15 portions of mulberry bark, 11 portions of earthworm, 9 portions of cyperus rotundus, and 7 portions of belamcanda chinensis.

3. A method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation, characterized in that, Includes the following steps: Horseradish, perilla leaves and fritillaria cirrhosa are crushed and filtered separately, then mixed to obtain material A. Anemarrhena asphodeloides, mulberry bark and earthworm are crushed and filtered separately, then mixed to obtain material B. Cynanchum paniculatum and Belamcanda chinensis are crushed and filtered separately, then mixed to obtain material C. After cleaning and removing impurities, materials A, B, and C are soaked in a 0.1%-0.3% sodium bicarbonate aqueous solution for 20-40 minutes, and then dried in a vacuum drying oven at 65-75℃ with a vacuum degree of 0.08-0.06 MPa. Add the pretreated material A to a dynamic extraction tank, add 10-15 times its weight of purified water, set the extraction temperature to 90-95℃, the stirring speed to 60-80 r / min, and extract for 3-4 hours. After filtration, obtain filtrate A. Add 8-10 times its weight of purified water to the filter residue and extract for another 1.5-2 hours. After filtration, obtain filtrate A. ∕ Combine filtrate A and filtrate A ∕ Extract A was obtained; Add 70%-70% ethanol aqueous solution to the pretreated material B, with a weight ratio of material B to ethanol aqueous solution of 1:8-12. Then add cellulase with an enzyme activity of 5000 U / g and pectinase with an enzyme activity of 3000 U / g, with a mass ratio of cellulase to pectinase of 1:

1. The amount of enzyme added is 0.5%-1% of the weight of material B. After enzymatic hydrolysis at 45-55℃ for 2-3 hours, place it in an ultrasonic extractor, set the power to 400-500W and the frequency to 35±5KHz, and extract for 1-1.5 hours. After filtration, extract B is obtained. Add the pretreated material C to a microwave extractor, add 6-8 times the weight of purified water, set the power to 600-700W, the temperature to 75-85℃, extract for 25-40 minutes, and filter to obtain extract C; Mix extract A, extract B and extract C, concentrate under reduced pressure at 50-60℃ to a relative density of 1.15-1.25 to obtain a concentrate, add 2-3 times its volume of anhydrous ethanol to the concentrate, stir and let stand for 12-18 hours, centrifuge and take the supernatant, recover the ethanol under reduced pressure until there is no alcohol odor, to obtain the crude extract. The crude extract was dissolved in purified water and loaded onto an HPD-100 macroporous resin column at a flow rate of 1-1.5 BV / h. After adsorption saturation, the column was eluted with 3-4 BV of purified water at a flow rate of 2-2.5 BV / h, followed by elution with 60%-70% ethanol aqueous solution at a flow rate of 1.5-2 BV / h for 4-5 BV. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain the alpine horseradish extract used for antitussive and anti-inflammatory purposes.

4. The method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation according to claim 3, characterized in that: Material A passes through a 100-120 mesh sieve, material B passes through an 80-100 mesh sieve, and material C passes through a 60-80 mesh sieve.

5. The method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation according to claim 3, characterized in that: The mass concentration of the ethanol aqueous solution is 75%, and the weight ratio of material B to the ethanol aqueous solution is 1:

10.

6. The method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation according to claim 3, characterized in that: The extraction parameters of the microwave extractor are 650W power, 80℃ temperature, and 30min extraction time.

7. The method for preparing an aqueous extract of horseradish for cough relief and anti-inflammation according to claim 3, characterized in that: The freeze-drying time is 24-36 hours.

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