WO2004064848A1 - Uses of naringenin, naringin and salts thereof as expectorants in the treatment of cough, and compositions thereof - Google Patents

Abstract

The invention relates to the use of Naringenin, Naringin and salts thereof in preparation of pharmaceutical compositions for the treatment of cough and phlegmatical conditions, to the uses of Naringenin, Naringin and salts thereof in the treatment of cough and phlegmatical conditions, and compositions thereof. The compositions of the invention contain effective amount of Naringenin, Naringin and salts thereof as active ingredients, and pharmaceutically acceptable carriers. The compositions of the invention have improving effects of treatment of cough and phlegmatical diseases, without side effects, and can be used to treat acute or chronic bronchitis, cold etc. relating cough and phlegmatical conditions.

Description

 Therapeutic use of naringenin, naringin and its salt for stopping phlegm and expectorant and pharmaceutical composition

 The invention relates to the field of treatment of cough and expectoration, in particular to the application of naringenin, naringin and its salt in the preparation of a pharmaceutical composition for cough and expectoration, and the preparation of naringenin, naringin and its salt Into a pharmaceutical composition, and a therapeutic use for stopping phlegm and expectorant. Background technique

 Cough, sputum, and asthma are common symptoms of respiratory diseases. They are usually caused by acute and chronic bronchitis and colds in daily life. Cough in the clinic is often accompanied by expectoration. Infants and middle-aged and elderly people are more likely to suffer from cough and sputum due to acute and chronic bronchitis and colds due to the influence of various factors such as physical fitness. As a common multiple disease, if the treatment is not well controlled at the beginning of the onset, but gradually develops, it can seriously affect people's health and quality of life.

 For the use of chemical drugs for cough and expectoration, cough medicines are now codeine, dextromethorphan hydrobromide C dextromethorphan> pentoxyveiine, benproperine, etc. ; French sputum drugs include ammonium chloride, acetylcysteine, bromhexine, etc. The existing antitussive and expectorant chemical medicines have one common disadvantage: none of the chemical medicines used for antitussives has an expectorant effect, and it has a better effect on sputum-free dry cough, but cannot be used to treat cough and sputum. Expectorant drugs are also ineffective in relieving cough. In addition, chemical drugs have strong side effects, such as codeine, which is prone to addiction; dextromethorphan hydrobromide can easily cause dizziness and belching; pentovirin can easily cause mild headache, dizziness, dry mouth, constipation, and also has Atropine-like effect, forbidden in glaucoma patients; benproperine is prone to dizziness, stomach burning and rash after taking it; ammonium chloride can have local irritating effects on gastric mucosa, ulcers and liver and kidney insufficiency Acetylcysteine has a special odor, which can cause nausea and vomiting. It is not suitable for patients with bronchitis because it is irritating to the respiratory tract. Bromohexyl bromide can cause stomach discomfort and is not suitable for those with peptic ulcers. patient.

 Many efforts have been made to use traditional Chinese medicine to relieve cough and expectorant. Traditional Chinese medicine compound preparations such as orange red pills, Chuanbei syrup and Xiaoyingchuantang are included in the Pharmacopoeia of the People's Republic of China (2000 edition) . However, in terms of its characteristics, the current traditional Chinese medicine for cough and expectoration has the disadvantage of taking a large amount of medicine, such as orange red pills, which are honey pills composed of 15 kinds of plant raw materials such as citrus red and tangerine peel. The amount of each dose is 10 grams. Above; Chuanbei syrup is a syrup made from the compound of four plant raw materials, Fritillaria chuanxiong, Platycodon grandiflora, ft. Leaf, and menthol. Xiaotongchuan syrup is a syrup made from plant material Manshanhong. Both of them take 10 ml at a time. None of the above three traditional Chinese medicine preparations is convenient for taking.

 Therefore, it is necessary to continue to develop new cough and expectorant drugs that are non-irritating to the stomach and respiratory tract, have small side effects, are convenient to carry and take, are safe, and have reliable curative effects and can be applied to both sputum-free dry cough and cough and sputum.

 Naringenin, chemical name is 5, 7, 4'-trihydroxydihydroflavone, is an aglycone of flavonoid compound naringin, which is widely present in plant material tissues such as flower buds of cherry blossoms and flower buds of plums. Flavonoids have the functions of anti-oxidation, anti-ulcer, inhibition of breast cancer proliferation, delayed breast tumor formation, and treatment of vascular diseases; naringenin is a derivative of naringenin. Combined with a glycoside formed by a disaccharide, it has been reported in the literature to be used in anti-oxidation, anti-inflammatory, anti-ulcer and other aspects. No naringenin, naringin and its salts have been reported to have cough and expectorant activity.

The molecular formula of naringenin is: C ₁₅ H ₁₂ 0 ₅

 The structure of naringenin is as follows:

柚皮素 (naringenin)

Naringenin

 1

Confirm this The molecular formula of naringin is: C ₂₇ H ₃₂ 0 ₁₄

 The structural formula of naringin is as follows:

 OH OH Naringin

 Since naringenin and naringin are flavonoids and have free phenolic hydroxyl groups, they can be formed with basic substances such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, etc. salt.

 Naringenin salt means naringenin sodium salt or naringenin salt produced by the reaction of naringenin with the above-mentioned alkali. Naringin salt refers to naringin sodium salt or naringin potassium salt produced by the reaction of naringin with the above-mentioned alkali. Summary of the Invention

 The object of the present invention is to provide a new therapeutic use of naringenin, naringin and its salts.

 The use of the present invention is: the application of naringenin, naringin and its salt in preparing a pharmaceutical composition for cough and expectorant.

 Another object of the present invention is to provide a pharmaceutical composition for relieving cough and expectorant.

 The pharmaceutical composition for cough and expectoration according to the present invention comprises an effective therapeutic dose of naringenin, naringin and its salt as active ingredients, and a pharmaceutically acceptable carrier.

 The effective therapeutic dose is 0.1-500 mg / kg body weight / day. The preferred daily dose is about 1 to 100 mg / kg body weight / day.

 The pharmaceutical composition is formulated into a dosage form selected from the group consisting of a tablet, a syrup, a capsule, a powder injection, an injection, and an inhaler.

 The pharmaceutical composition can be used for oral administration, injection administration, or pulmonary inhalation administration.

 In the pharmaceutical composition, the method for preparing naringenin, naringin and its salt is as follows:

 Naringin can be based on Rosenmund (Rosenmund, Ber., 61, 2608 (1958)) and Zemlen, Bognar

(Ber., 75, 648 (1942)).

 Naringin can also be obtained by extracting plant raw materials.

 Naringin-containing plant raw materials include coriander shell, coriander, citrus, lemon, grapefruit, tangerine, orange and the like.

 The method for extracting naringin includes the following steps: pulverizing the naringin-containing plant raw material, extracting one to three times with water, filtering, and combining the filtrates; concentrating the filtrate and performing alcohol precipitation, filtering, and recycling the filtrate to ethanol or directly applying the filtrate The column was subjected to column separation, and the organic solvent was eluted. After the solvent was recovered and removed from the eluent, the crude naringin was obtained, and then recrystallized from one to ten times to obtain a naringin monomer.

 There are three ways to prepare naringenin:

 In the first method, naringin is hydrolyzed to obtain a naringenin monomer.

 The method for preparing naringenin includes the following steps: dissolving naringin in water, stirring uniformly, adding acid or alkali or enzymatic hydrolysis, filtering through precipitation to obtain a crude naringenin, and then recrystallizing from an organic solvent one to ten times to obtain pomelo Cortexin monomer.

 The enzyme is glucuronidase or other enzymes that can cleave six carbon sugars.

 The second method is to extract naringenin from plant raw materials containing naringenin.

Naringenin-containing plant materials include flower buds of cherry blossoms and flower buds of plums. The naringenin extraction method includes the following steps: pulverizing the naringenin-containing plant raw material, extracting it with an organic solvent one to three times, filtering, and combining the filtrates; the filtrate is concentrated into an extract, and the extract precipitates to obtain a crude naringenin; After one to ten recrystallizations from an organic solvent, a naringenin monomer is obtained.

 The third method is to directly extract naringenin from the naringin-containing plant raw material by extraction.

 The naringenin extraction method includes the following steps: crushing the naringin-containing plant material, extracting it with water or an organic solvent one to three times, filtering, and combining the filtrates; the filtrate is concentrated into an extract, the extract is dissolved in water or an organic solvent, and an acid or Alkaline or enzymatic hydrolysis, filtration, to obtain crude naringenin; and recrystallization from one to ten times to obtain naringenin monomer.

 The enzyme is glucuronidase or other enzymes that can cleave six carbon sugars.

The organic solvent used in the naringin and naringen extraction method is selected from the group consisting of ethanol, methanol, chloroform, ethyl acetate, and petroleum ether. The naringenin salt is a metal salt formed by the reaction of naringenin and a corresponding alkaline substance, and the molecular formula is: C ₁₅ H ( ₁₂ .. _N ) 0 ₅ Y _n , n = 1-3, Y is a metal ion .

 Naringenin salt is prepared by the following methods: 0 ~ 10 (under TC condition, naringenin reacts with the corresponding alkaline substance to form the corresponding naringenin metal salt; during the reaction, naringenin and alkaline The molar ratio of the substances is 1: 1 to 1: 3.

The alkaline substance is selected from one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate, and the naringenin salt obtained by the reaction is the sodium or potassium salt of amaginin. A salt has a molecular formula of: C ₁₅ H ( _{12. N} ) 0 ₅ Y _n , n = 1-3, and Y is a K ion or Na ion.

The naringin salt is a metal salt formed by the reaction of naringin with a corresponding basic substance, and the molecular formula is: C ₂₇ H ( _3¾l ) 0 ₁₄ Y _n , n = 1-2, and Y is a metal ion.

 Naringin salt is prepared by the following method: Under the condition of 0 ~ 40Ό, naringin reacts with the corresponding basic substance to form a metal salt of naringin; during the reaction, the mole of naringin and the basic substance The ratio is 1: 1 to 1: 2.

The alkaline substance is selected from one of sodium hydroxide, hydrogen, potassium oxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate, and the naringenin salt obtained by the reaction is the sodium salt of naringin or Potassium salt, its molecular formula is: C ₂₇ H ( _{32. N} ) 0 ₁₄ Y _n , n = 1-2, Y is K ion or Na ion.

 Another object of the present invention is to provide a method for treating cough and expectorant.

 The treatment method for relieving cough and expectorant according to the present invention comprises administering a pharmaceutical composition as described above to a lactating animal, especially to a human. Naringenin is a trihydroxydihydroflavonoid compound, which is stable in nature and exists as the original drug after being inserted into the human body; naringenin is a derivative of naringenin and is composed of naringenin at position 7 After entering a human body, a glycoside formed by disaccharides is hydrolyzed under the action of gastric acid, so it mainly functions in the form of naringenin in the human body, and a small amount exists in the form of naringin because of incomplete hydrolysis. After entering into the human body, naringenin salt is converted into naringenin by the action of gastric acid, thereby exerting its effect. After entering the human body, naringin salt is first converted into naringin under the action of gastric acid, and then hydrolyzed by gastric acid to produce naringenin and play a role. The present invention confirms through pharmacological and pharmacodynamic experiments that naringenin, naringin and its salts have good cough and expectorant effects, and has obvious curative effects on cough and sputum caused by acute and chronic bronchitis and colds.

 The inventors carried out an antitussive experiment on naringenin, naringin and its salts in experimental mice. The results showed that the tolerance time of naringenin, naringin and its salts on cough induced by irritated mice was significantly prolonged compared with the blank control group, and there were statistically significant differences; compared with the positive control drug hydrogen Compared with dextromethorphan bromate, the tolerance time was prolonged, and the effect was more significant than that of dextromethorphan hydrobromide. It shows that naringenin, naringin and its salt have good anti-snoring effect.

The inventors carried out an expectoration experiment of experimental animals on mice with naringenin, naringin and its salts. The results showed that naringenin, naringin and its salt increased the bronchial secretion of mice, compared with the blank control group, there was a significant increase, and there was a statistically significant difference. Compared with the positive control drug Tantanjing, the bronchial secretion of mice has a significant increase, there is a statistically significant difference, and the effect is significantly higher than that of the positive drug Tantanin. It shows that naringenin, naringin and its salt have expectorant effects. The experiment of the present invention proves that naringenin, naringin and its salt not only have good antitussive and expectorant effects, but also do not show toxicity in mouse and animal experiments. Animal experiments show that when naringenin or its salt is administered orally at a dose of 6 g / kg, there is no toxic reaction in the animal, which is equivalent to 460 times the normal human dose; when 3 g / kg of naringin or When the salt was orally administered to animals, no toxic reaction was seen in the animals, and the dose was equivalent to 230 times the normal human dose.

 In summary, it is shown that naringenin, naringin and its salt have good cough and expectorant effects, no toxic and side effects are seen, and can effectively treat cough and sputum caused by acute and chronic bronchitis and colds in the clinic. . Therefore, it can be used for preparing antitussive and expectorant drugs.

 The pharmaceutical composition prepared by naringenin, naringin and salts thereof has good cough and expectorant effects, no toxic and side effects, and can effectively treat cough and sputum caused by acute and chronic bronchitis, colds, etc. Many symptoms, and has the characteristics of stable quality, small doses, rapid efficacy and so on. detailed description

 The present invention will be further described with reference to the following embodiments.

 The solids in the solid mixtures, liquids in liquids, and percentages of solids in liquids in the examples are calculated in wt / wt, vol / vol, wt / vol, respectively, unless otherwise stated. Example 1: Preparation of Naringin Monomer

 Cut the husk plant raw material into cut pieces, add 100 没 water to the plant raw material, and extract the water three times, each time for 1 hour, and filter; the filtrate is concentrated into an extract, passed through a macroporous resin column, first eluted with water, discard the water solution, and then Eluted with 70% ethanol, collected the ethanol eluate, recovered ethanol to obtain a precipitate, and recrystallized three times with water to obtain a naringin monomer. The transfer rate is 90.7%. Example 2: Preparation of Naringin Monomer

 The citrus plant material is crushed, but sieve, add 10CTC water to the plant material, extract twice, filter for 1 hour each time, and combine the filtrates; concentrate the filtrate, add ice-free ethanol to 30% ethanol concentration, leave it overnight, and filter The supernatant was recovered ethanol. The extract was put on a polyamide column, first eluted with water, the aqueous solution was discarded, and then eluted with 80% ethanol. The ethanol eluate was collected and ethanol was recovered to obtain a precipitate. After recrystallization with anhydrous ethanol five times, pomelo peel was obtained. Glycomonomer. The transfer rate was 87.4%. Example 3: Preparation of Naringin Monomer

 The orange-red plant raw material was cut into pieces, which were extracted twice with 70 ° C water for 3 hours each time, filtered, and the filtrate was combined; the filtrate was concentrated into an extract, and 95% ethanol was added to the extract to 60% ethanol concentration, and it was left to stand overnight A precipitate is generated, filtered, depreciated, and the filtrate is recovered to ethanol. The obtained liquid is passed through a polyamide column, first eluted with water, the aqueous solution is discarded, and then 80% ethanol is collected; the ethanol eluate is collected, recovered and concentrated to obtain a precipitate. , Recrystallized four times with anhydrous ethanol to obtain naringin monomer. The transfer rate was 89.7%. Example 4: Preparation of Naringin Monomer

 The lemon plant raw material was not pulverized, and it was extracted three times with ultrasonic water for 1 hour each time, filtered, and the filtrates were combined; concentrated to obtain an extract, and the extract was directly passed through a polyamide column, which was first eluted with water, and the aqueous solution was discarded. % Ethanol elution; collecting the ethanol eluate, recovering the ethanol, and filtering to obtain a precipitate. The precipitate was recrystallized once with ethyl acetate to obtain a naringin monomer. The transfer rate was 85.3%. Example 5: Preparation of Naringin Monomer

 The raw plant material is crushed, sieved through a 20-mesh sieve, and extracted three times at room temperature with water, filtered for two days each time, and the filtrates are combined; the filtrate is added with 95% ethanol to an ethanol concentration of 50%, and the liquid is allowed to stand. Recover ethanol from the solution, dissolve it with water, and pass through the dextran gel column. Elute with water first, discard the water, and then elute with 40% ethanol. Collect the ethanol eluate, recover the ethanol, and filter to obtain a precipitate. The ester was recrystallized ten times to obtain a naringin monomer. The transfer rate was 92.1%. Example 6: Preparation of Naringin Monomer

Grapefruit plant raw materials are cut into pieces, 10CTC water is added to the plant raw materials, and extracted twice, each time for 2 hours, filtered, and the filtrates are combined; The filtrate was concentrated to obtain an extract. The extract was passed through a macroporous resin column, first eluted with water, discarded the aqueous solution, and then eluted with 60% ethanol. The ethanol eluate was collected, ethanol was recovered, and filtered to obtain a precipitate. Recrystallize three times with water to obtain naringin monomer. The transfer rate was 87.6%. Example 7: Preparation of naringenin monomer

 Naringin (purity 98.2%), add 10 times water, mix well, add hydrochloric acid, adjust pH = 1, mix well; heat at 100Ό for 2 hours; after filtration, filter to obtain crude naringenin; After recrystallization from water ethanol five times, naringenin monomer was obtained. The transfer rate was 91.4%. Example 8: Preparation of Naringenin Monomer

 Naringin (purity 98.2%), add 20 times water, mix well, add sulfuric acid, adjust pH = 0, mix well; 8CTC heats and hydrolyzes for 3 hours; after the hydrolysis, filter to obtain crude naringenin; use ethyl acetate After the ester was recrystallized ten times, naringenin monomer was obtained. The transfer rate was 89.3%. Example 9: Preparation of Naringenin Monomer

 Naringin (purity 98.2%), add 50 times water, mix evenly, add sodium hydroxide solution, adjust pH = 13, mix well;

Hydrolysis at 80 ° C for 1.5 hours. After the hydrolysis, adjust the pH to 1 with hydrochloric acid and filter to obtain the crude naringenin. After recrystallization with acetone once, the naringenin monomer was obtained. The transfer rate was 86.9%. Example 10: Preparation of Naringenin Monomer

 Naringin monomer (purity 98.2%), add 35 times water, mix and mix, add glucuronidase to make the enzyme concentration in the sample to be

0.1 mmol / L, mix evenly; heat hydrolysis in a 37Ό water bath for 8 hours; after the hydrolysis is completed, filter to obtain a crude naringenin; and recrystallize four times with anhydrous ethanol to obtain a naringenin monomer. The transfer rate was 94.8%. Example 11: Preparation of Naringenin Monomer

 Sakura flower buds, plant material was added with absolute ethanol, extracted three times, the extraction temperature was 5CTC, 1 hour each time, filtered; the filtrate was concentrated into extracts, precipitated, and filtered to obtain crude naringenin; acetic acid was used for precipitation The ethyl ester was crystallized once to obtain a naringenin monomer. The transfer rate is 90.7%. Example 12: Preparation of Naringenin Monomer

 Flower buds of plum, acetone-free plant material, immersed in 25Ό, extracted twice, 48 hours each time, filtered, combined with filtrate; the filtrate was concentrated into an extract and left overnight to obtain a precipitate, and the precipitate was dried to obtain a crude naringenin ; After recrystallizing five times with chloroform, naringenin monomer is obtained. The transfer rate was 87.4%. Embodiment 13: Preparation of Naringenin Monomer

 The flower buds of the plum were added with ethyl acetate without plant material, soaked at 20 ° C, extracted once for 72 hours each time, filtered, and the filtrate was combined; the filtrate was concentrated into an extract and left overnight to obtain a precipitate, and the precipitate was dried to obtain pomelo peel Crude product; after recrystallizing ten times with acetone, naringenin monomer was obtained. The transfer rate was 85.6%. Example Fourteen: Preparation of Naringenin Monomer

 The raw plant material is crushed, passed through a 20-mesh sieve, and extracted three times with water at room temperature, filtered for two days each time, and the filtrates are combined; the filtrate is concentrated, and hydrochloric acid is added to make the liquid pH = 1, and the solution is heated at 90 ° C for hydrolysis. After that, a precipitate was obtained, and the precipitate was dried to obtain a crude naringenin; the precipitate was recrystallized four times with ethyl acetate to obtain a naringenin monomer. The transfer rate is 90.7%. Example 15: Preparation of Naringenin Monomer

Cut grapefruit raw materials into pieces, add 100 ° C water without plant raw materials, extract once, 2 hours each time, filter, and combine the filtrates; concentrate the filtrate, add sodium hydroxide to the solution pH = 11, 70 ° C, heat and hydrolyze, and hydrolyze At the end, add hydrochloric acid to adjust pH = 1, and filter to obtain a precipitate. The precipitate was dried to obtain a crude naringenin; the precipitation was recrystallized three times with acetone to obtain a naringenin monomer. The transfer rate was 89.2%. Example 16: Preparation of Naringenin Monomer

 The orange-red plant material was not crushed, and extracted three times with acetone for 24 hours each time, filtered, and the filtrates were combined; concentrated to obtain an extract, the extract was dissolved by adding 30% ethanol, and hydrochloric acid was added to make the liquid pH = 2, and heated at 95 ° C for hydrolysis At the end of the hydrolysis, filtration was performed to obtain a precipitate, and the precipitate was dried to obtain a crude naringenin; the precipitation was recrystallized five times with chloroform to obtain a naringenin monomer. The transfer rate was 87.7%. Example 17: Preparation of Naringenin Monomer

 Cut husk plant material into cut pieces, add 100 milliliters of water to the plant material, add concentrated hydrochloric acid to make the solution pH = 2.5, and extract three times for 1.5 hours each time, filter, and combine the filtrates; concentrate the filtrate to obtain an extract, and leave the extract overnight Precipitation was obtained; the precipitate was separated, dissolved with acetone, filtered, and the precipitate was discarded. The filtrate was recovered with acetone to obtain a precipitate, which was dried to obtain a crude naringenin; the precipitate was recrystallized three times with chloroform to obtain a naringenin monomer. The transfer rate was 90.4%. Example 18: Preparation of Naringenin Monomer

 Shatian pomelo plant raw material was crushed, 12 times 10CTC water was added to the plant raw material, potassium hydroxide was added to make the solution pH = 14, and the extraction was performed twice for 45 hours, filtered, and the filtrates were combined; the filtrate was concentrated to obtain an extract, and the extract was placed Overnight, a precipitate was obtained; the precipitate was separated, dissolved in anhydrous ethanol, filtered, the precipitate was discarded, and the filtrate was recovered to obtain ethanol, and the precipitate was dried, to obtain a crude naringenin; the precipitate was recrystallized four times with acetone, to obtain a naringenin monomer . The transfer rate was 88.3%. Example 19: Preparation of naringenin salt

 Under 0Ό conditions, take 1 mole of naringenin to dissolve in ethanol, take 1 mole of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringenin ethanol solution, stir for 2 hours, and filter to obtain grapefruit. Sodium salt of cortex.

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate may be used instead of sodium hydroxide to obtain the potassium salt of naringenin; the same equivalent of sodium bicarbonate or sodium carbonate may be used instead of sodium hydroxide, that is, Sodium naringenin is obtained. Example 20: Preparation of naringenin salt

 At 20Ό, take 1 mole of naringenin to dissolve in ethanol, 3 moles of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringenin ethanol solution, stir for 2 hours, and filter to obtain pomelo Sodium salt of cortex. .

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate can be used instead of sodium hydroxide to obtain the potassium salt of naringenin; or the same equivalent of sodium bicarbonate or sodium carbonate is used instead of sodium hydroxide to obtain Sodium salt of naringenin. Example 21: Preparation of naringenin salt

 Under 5CTC conditions, take 1 mole of naringenin to dissolve in ethanol, take 2 moles of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringenin ethanol solution, stir for 2 hours, and filter to obtain pomelo Sodium salt of cortex.

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate may be used instead of sodium hydroxide to obtain the potassium salt of naringenin; the same equivalent of sodium bicarbonate or sodium carbonate may be used instead of sodium hydroxide, that is, Sodium naringenin is obtained. Example 22: Preparation of naringenin salt

 Under 10CTC conditions, take 1 mole of naringenin to dissolve in ethanol, take 3 moles of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringenin ethanol solution, stir for 2 hours, and filter to obtain grapefruit. Sodium salt of cortex.

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate may be used instead of sodium hydroxide to obtain the potassium salt of naringenin; the same equivalent of sodium bicarbonate or sodium carbonate may be used instead of sodium hydroxide, that is, Sodium naringenin is obtained. Example 23: Preparation of Naringin

At 0 ° C, take 1 mole of naringin to dissolve in ethanol, take 1 mole of sodium hydroxide to dissolve in water, and slowly add the sodium hydroxide aqueous solution. Add naringin ethanol solution, stir for 2 hours, and filter to obtain the sodium salt of naringin.

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate may be used instead of sodium hydroxide to obtain the potassium salt of naringin; the same equivalent of sodium bicarbonate or sodium carbonate may be used instead of sodium hydroxide, that is, Sodium naringin is obtained. Example 24: Preparation of Naringin

 At 20 ° C, take 1 mole of naringin to dissolve in ethanol, take 2 moles of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringin ethanol solution, stir for 2 hours, and filter. Sodium naringin is obtained.

 In this embodiment, the same equivalent of potassium hydroxide, potassium bicarbonate, or potassium carbonate can be used instead of sodium hydroxide, and the potassium salt of naringin can also be replaced by the same equivalent of sodium bicarbonate or sodium carbonate. Sodium salt of naringin. Example 25: Preparation of Naringin

 At 40 ° C, take 1 mole of naringin to dissolve in ethanol, take 2 moles of sodium hydroxide to dissolve in water, slowly add the sodium hydroxide aqueous solution to the naringin ethanol solution, stir for 2 hours, and filter. Sodium naringin is obtained.

 In this embodiment, the same equivalent of potassium hydroxide or potassium bicarbonate or potassium carbonate may be used instead of sodium hydroxide to obtain the potassium salt of naringin; the same equivalent of sodium bicarbonate or sodium carbonate may be used instead of sodium hydroxide, that is, Sodium naringin is obtained. Example 26: Determination of Naringenin monomer purity

 Take an appropriate amount of naringenin monomer prepared by the above extraction method, dissolve it in methanol and make it into a volumetric flask, filter it with a microporous membrane (0.45um), and inject it into a high-performance liquid chromatography. The provided naringenin reference substance was quantified by an external standard method to obtain the naringenin content in the naringenin sample obtained by extraction. The results are shown in Table 1.

Chromatographic conditions: Agilent 1100 HPLC (autosampler, vacuum degasser, quaternary pump, column oven, diode array detector); Chromatographic column: MARKER ODS column (5um, 4.0 X 250mm); mobile phase : Methanol-water (50:50); detection wavelength: 288nm; column temperature 30 ° C _; flow rate: 1 ml / min; injection volume: 5μΙ.

It can be seen from the table results that the naringenin content in the extracted samples is higher than 95%, indicating that the naringenin sample obtained by the extraction method of the present invention has high purity. Example twenty-seven naringin monomer purity determination experiment

 Take an appropriate amount of naringin monomer prepared by the above extraction method, dissolve it in methanol and make it into a volumetric flask, filter it with a microporous membrane (0.45um), and then inject it into a high-performance liquid chromatography. The provided naringin reference substance was quantified by an external standard method, and the content of naringin monomer in the naringin sample was obtained. The results are shown in Table 2.

Chromatographic conditions: Agnent1100 high performance liquid chromatograph (autosampler, vacuum degasser, quaternary pump, column oven, diode array detector); chromatographic column: MARKER ODS column (5um, 4.0X 250mm) _; mobile phase : Acetonitrile-0.05M KH ₂ P0 ₄ buffer (20: 80); detection wavelength: 283nm; column temperature 3CTC; flow rate: 1 ml / min; injection volume: 5μΙ. Table 2: Sample test results

从表结果看出，提取所得样品中柚皮苷单体的含量均高于 95%。说明本发明的提取方法所得到的 柚皮苷样品纯度高。 综上所述， 说明本发明的柚皮苷和柚皮素提取方法， 能有效地从含柚皮苷植物原料中提取得到柚 皮苷单体， 能有效的通过将柚皮苷粗品 /单体水解成柚皮素得到柚皮素单体， 能有效地从含柚皮素 /柚皮 苷植物原料中提取得到柚皮素单体， 所得柚皮苷样品和柚皮素样品纯度高， 含量均高于 95%以上。 工 艺简单可行。 实施例二十八： 柚皮苷药物的止晐药理学实验 -

It can be seen from the table results that the naringin monomer content in the extracted samples is higher than 95%. This shows that the naringin sample obtained by the extraction method of the present invention has high purity. In summary, the method for extracting naringin and naringenin according to the present invention can effectively extract the naringin monomer from the naringin-containing plant raw material, and can effectively use the naringin crude product / monomer. Hydrolyzed into naringenin to obtain naringenin monomer, which can effectively extract naringenin from naringenin / naringenin-containing plant raw materials. The naringin sample and naringenin sample are high in purity and content. Above 95%. The process is simple and feasible. Example 28: Pharmacological Experiments of Naringin Drugs

1. Experimental animals: NIH mice, males, weighing 18.2-21.7g, common grade standard, 130 in total. The animals were weighed, numbered, and 120 healthy mice weighing 18.5-21.0 g were selected. Sorted by weight, divided into eight groups by random grouping, 15 in each group. The negative control group, the positive control group, and the naringin high, middle, and low naringin sodium salt high, middle, and low drug sample groups were set.

 2. Sample source and treatment-1) Blank control group: saline, 0.9% NaCI content.

 2) Positive control group: Dissolve 30 mg of dextromethorphan hydrobromide in 20 ml of physiological saline to obtain a positive control solution of dextromethorphan hydrobromide. The concentration of dextromethorphan hydrobromide is 1.5 mg / ml.

 3) Low-dose group of naringin: Take an appropriate amount of naringin, and dilute it in a volumetric flask with physiological saline. The concentration of naringin is 0.5 mg / ml.

 4) Medium dose group of naringin sample: Take an appropriate amount of naringin, and dilute it in a volumetric flask with physiological saline, and the naringin concentration is 1.5 mg / ml.

 5) Naringin sample 髙 Dosage group: Take an appropriate amount of naringin and dilute it in a volumetric flask with physiological saline, and the naringin concentration is 4.5 mg / ml.

 6) Low-dose group of naringin sodium salt: Take an appropriate amount of naringin sodium salt and dilute it in a volumetric flask with physiological saline. The concentration of naringin sodium salt is 0.5mg / ml. '

7) Medium dose group of naringin sodium salt sample: Take an appropriate amount of naringin sodium salt sample, make up to volume with physiological saline, and set the concentration of naringin sodium salt at 1.5mg / ml. 8) High-dose group of naringin sodium salt: Take an appropriate amount of naringin sodium salt sample and dilute to volume with physiological saline. The concentration of naringin sodium salt is 4.5mg / ml.

 The naringin sample used was a naringin monomer extracted from the plant raw material according to the method described above, and the purity was above 95%.

 3. Experimental method: (Concentrated ammonia spray method)

 Mice were administrated for 1 hour and started to receive spray. Spray the aerosol of concentrated ammonia water for a certain period of time. After the spray is over, remove the mice immediately and observe the cough response. Observe the number of coughs in one minute. If there are more than three typical coughing movements in one minute (constriction or contraction of the abdominal muscles, open mouth at the same time, and sometimes cough), it is counted as "cough". Otherwise, it counts as "no choking".

 4. Experimental process: '

Sequential method (up and down method) was used to determine the spray time (EDT _5Q ) that caused cough in half of the mice. Calculate the R value. If the R value is greater than 130%, the drug has an antitussive effect. If the R value is greater than 150%, it indicates a significant antitussive effect. Calculated as follows:

EDT _{50 in the} administration group

R ⁼ EDT _{5 of the} control group. ^X Lake%

EDT ₅₀ = log- ¹ c / n (where n is the number of animals, c is the sum of rx values, r is the number of animals per dose group, and x is the logarithm of the dose (ie, spray time).) '

5. Experimental results:

 According to statistics, half of the cough time and antitussive effect of each sample group are shown in Table 3.

 Comparing the tolerance time of naringin samples and the positive control drug dextromethorphan hydrobromide to irritated mice caused by cough, the naringin has a significantly longer tolerance time in mice than the positive control. The drug dextromethorphan hydrobromide has a long tolerance time and is statistically significant. This shows that naringin's tolerance time to cough induced by irritation in mice is longer than that of dextromethorphan hydrobromide, the tolerance time is prolonged, and the antitussive effect is good. Example 29: Expectorant Pharmacology Experiment of Naringin Drugs

 1. Experimental animals: NIH mice, female, «18.8-22.7g, clean grade standard, a total of 90 animals. The animals were weighed, numbered, and 80 healthy mice weighing 19.1-22.3 grams were selected. Sorted by weight, divided into eight groups by random grouping, 10 in each group. Negative control, positive control and naringin high, medium and low, naringin sodium salt high, medium and low dose groups. All were administered orally at a dose of 10ml / kg body weight.

 2. Sample source and processing:

 1) Blank control group: saline, 0.9% NaCI.

 2) Positive control group: 0.2g of Tankejing powder was dissolved in 10ml of physiological saline to obtain the positive control Tankejing solution at a concentration of 20mg / ml.

3) Naringin sample low-dose group: Precisely weigh 5 mg of naringin sample, make up to 10 ml volumetric flask with physiological saline, dissolve it with ultrasound for 5 minutes, and shake to obtain. The concentration was 0.5 mg / ml. 4) Mid-dose group of naringin sample: 15 mg of naringin sample is accurately weighed, and the volume is adjusted into a 10 ml volumetric flask with physiological saline. The ultrasonic dissolution is performed for 5 minutes, and the hook is shaken to obtain. The concentration was 1.5 mg / ml.

 5) Naringin sample high-dose group: Precisely weigh 45 mg of naringin sample, make up to a 10 ml volumetric flask with physiological saline, dissolve it with ultrasonic waves for 5 minutes, and shake the hook to obtain. The concentration was 4.5 mg / ml.

 6) Low-dose group of naringin sodium salt: Precisely weigh 5mg of naringin sodium salt sample, make up to 10ml volumetric flask with physiological saline, dissolve by ultrasonic for 5 minutes, and shake to obtain. The concentration was 0.5 mg / ml.

 7) Medium dose group of naringin sodium salt sample: Weigh 15mg of naringin sodium salt sample accurately, make up to a 10ml volumetric flask with physiological saline, dissolve it with ultrasound for 5 minutes, and shake to obtain. The concentration was 1.5 mg / ml.

 8) High-dose group of naringin sodium salt: Precision weigh 45 mg of naringin sodium salt sample, dilute to volume in a 10 ml volumetric flask with normal saline water, dissolve by ultrasonic for 5 minutes, and shake to obtain. The concentration was 4.5 mg / ml.

 3. Experimental method:

 1) Drawing of standard phenol red curve: Weigh out a certain amount of phenol red with an analytical balance, dissolve it with 5% sodium bicarbonate, make up 12.5ng per 1ml, and then sequentially dilute to phenol red per ml 6.25ng, 3.125ng, 1.5625ng, 0.7813ng, 0.3906ng, 0.1953ng, 0.0977ng, 0.0488ng, OD value was measured by spectrophotometer. Using the phenol red concentration as the ordinate and the OD value as the abscissa, the regression equation was calculated based on the phenol red concentration and the OD value. The excretion of each phenol red was calculated according to the regression equation.

 2) Mice fast for 12 hours.

 3) Oral administration. In the order of the animal number, each mouse was stopped for 3 minutes after gavage, and then the other was given at an interval of 3 minutes. The total gavage time for each group of 10 mice was 30 minutes.

 4) 0.5 hours after intragastric administration of each rat, 0.2ml 5% phenol red physiological saline solution was injected intraperitoneally. In order, each mouse was injected intraperitoneally 3 minutes after phenol red, and then the other one was injected for 10 mice for a total of 30 minutes.

 5) 0.5 hours after intraperitoneal injection of each mouse, the mice were sacrificed in order to sacrifice the cervical spine, and the sacrifice interval was 3 minutes. After the animals were sacrificed, they were fixed on the operation board in the supine position, the skin in the middle of the neck was cut, the trachea was separated, and the trachea was supported with small forceps.

 6) Use a large syringe to absorb the saline solution to rinse the outer wall of the trachea, wash off the blood and the phenol red in the outer wall of the trachea, and filter the paper to absorb the dry cleaning solution.

 7) Cut the trachea before the branch of the trachea, then cut the trachea from the upper end of the other side of the thyroid cartilage (including the circular thyroid cartilage).

8) Put each trachea segment into a test tube containing 1.5ml of 5% NaHC0 ₃ solution in advance.

 9) Complete the tracheal separation and shearing work within 3 minutes. The second mouse was treated the same way. The method is as above.

 10) Put each test tube on an ultrasonic cleaner for 5 minutes to release the phenol red in the trachea section.

 11) The OD value of the solution in each test tube was measured at 546 nm of a 721 spectrophotometer.

 12) Leave the test tube containing the trachea section overnight, and re-measure the OD value after 24 hours.

 13) Calculate the content of phenol red according to the regression equation. Calculation formula: Y = 7.6266X—0.0554. X is OD value, Y is phenol red

14) Calculate the corrected phenol red content based on the phenol red content and animal weight, and perform analysis of variance using SPSS 8.0 statistical software ₍ corrected phenol red content = phenol red content (ng) / mouse weight (kg)

 4. Experimental results:

 According to statistics, the phenol red excretion amount of each dose group is shown in Table 4 below.

 The expectorant effect of hesperidin (x ± s)

 Group

Dose animal tracheol corrected phenol red content ^a expectoration rate ^b

 P value

Code Sample name (mg / kg) Red content (ng) (ng / kg) (%)

1 saline 0 0.3031 ± 0.0747 15.4576 + 4.0701

 45.0285 + 21.8238 P <

 2 Phlegm and cough net 200 0.8621 ± 0.3981 291.30

 0.01

 25.5811 ± 11.8338 P <

 3 Naringin sample low dose group 5 0.4914 + 0.2215 159.02

0.05 26.7597+ 13.1502 ρ <Naringin sample in middle dose group 15 0.5296 ± 0.2195 173.12

 0.01

 64.4162 + 18.7239 Ρ <

 Naringin sample high-dose group 45 1.1488 ± 0.2431 416.72

 0.01

 Naringin sodium salt sample 26.1805 + 6.9069 Ρ <

 5 0.5250 ± 0.1265 169.37 Low-dose group 0.05 Naringin sodium salt sample 29.3153 ± 7.7189 P <

 15 0 · 5748 ± 0 · 1417 189.65 Medium dose group 0.01 Naringin sodium salt sample 66.8867 ± 17.6117 Ρ <

 45 1 · 3115 ± 0 · 3232 432.71 High-dose group 0.01 a: Corrected phenol red content = phenol red content / animal weight

 b: expectoration rate = dosing group / blank control group X 100%

 Phenol red content calculation method (ng): OD value X 7.6266 From the naringin sample and the positive drug Tankejing, oral administration to increase the bronchial secretion of mice, the naringin was low, medium and high dose groups. Compared with the blank control group, the secretion of mouse bronchial secretion in the dose group had a significant increase, and there was a statistically significant difference. Compared with the positive control drug Tankejing in the high-dose group, the bronchial secretion of mice was significantly increased, and there was a statistically significant difference, and the effect was significantly higher than that of the positive drug Tankejing. This shows that naringin has a good expectorant effect. Example Thirty: Pharmacological Experiments of Naringin Drugs

 1. Experimental animals: NIH mice, male, weighing 19.3-22.6g, common grade standard, 130 in total. The animals were weighed, numbered, and 120 healthy mice weighing 19.5-22.1 g were selected. Sorted by weight, divided into eight groups by random grouping, 15 in each group. Negative control group, positive control group and naringenin drug sample low-dose group, naringenin drug sample medium-dose group, naringenin drug sample high-dose group, naringenin sodium drug sample low-dose group, naringenin Sodium salt drug samples in the high-dose group, naringenin sodium salt drug samples in the high-dose group.

 2. Sample source and processing: ''

 1) Blank control group: saline, 0.9% NaCI.

 2) Positive control group: Dissolve 30 mg of dextromethorphan hydrobromide in 20 ml of physiological saline to obtain a positive control solution of dextromethorphan hydrobromide. The concentration of dextromethorphan hydrobromide is 1.5 mg / ml.

 3) Low-dose group of naringenin: Take an appropriate amount of naringenin and dilute in a volumetric flask with physiological saline, and the concentration of naringenin is 0.5 mg / ml.

 4) Medium dose group of naringenin: Take an appropriate amount of naringenin and dilute it in a volumetric flask with physiological saline, and the concentration of naringenin is 1.5mg / ml.

 5) High-dose group of naringenin: Take an appropriate amount of naringenin and dilute it in a volumetric flask with physiological saline, and the naringenin concentration is 4.5 mg / ml.

 6) Low-dose group of naringenin sodium salt: Take an appropriate amount of naringenin sodium salt and dilute to volume with physiological saline. The concentration of naringenin sodium salt is 0.5mg / ml.

 7) Dosage group of naringenin sodium salt: Take an appropriate amount of naringenin sodium salt and dilute it in a volumetric flask with physiological saline. The concentration of naringenin sodium salt is 1.5mg / ml.

 8) High-dose group of naringenin sodium salt: Take an appropriate amount of naringenin sodium salt and make up to volume with physiological saline. The concentration of naringenin sodium salt is 4.5mg / ml.

 3. Experimental method: (Concentrated ammonia spray method)

 One hour after the mice were gavage, they started to receive spray. Spray the aerosol of concentrated ammonia water for a certain period of time. After the spray is over, remove the mice immediately and observe the cough response. Observe the number of coughs in one minute. If there are more than three typical coughing movements in one minute (constriction of the abdominal muscles or contraction of the chest, open mouth at the same time, and sometimes snoring), it is counted as "coughing". Otherwise it counts as "no cough".

 4. Experimental process:

Sequential method (up and down method) was used to determine the spray time (EDT _{5 ()} ) of coughing in half of the mice from bow I. Calculate the R value. If the R value is greater than 130%, the drug has an antitussive effect. If the R value is greater than 150%, it indicates that there is a significant effect of stopping the maggot. Calculated as follows: EDT50 of the administration group

 R = X 100%

 EDT50 in control group

EDT ₅₀ = log- ¹ c / n (where n is the number of animals, c is the sum of rx values, r is the number of animals per dose group, and x is the logarithm of the dose (ie, spray time).)

 5. Experimental results-According to statistics, half of the cough time and antitussive effect of each sample group are shown in Table 5. Table 5. Antitussive effect of each sample

从柚皮素样品和阳性对照药物氢溴酸右美沙芬， 经口给药对刺激小鼠引起咳嗽的耐受时间比较， 柚皮素对小鼠耐受时间有显著的延长， 比阳性对照药品氢溴酸右美沙芬的耐受时间长， 在统计学上有 显著差异。 从柚皮素钠盐样品和阳性对照药物氢溴酸右美沙芬， 经口给药对剌激小鼠引起咳嗽的耐受 时间比较， 柚皮素钠盐对小鼠耐受时间有显著的延长， 比阳性对照药品氢溴酸右美沙芬的耐受时间长， 在统计学上有显著差异。 说明柚皮素、 柚皮素盐对剌激小鼠引起咳嗽的耐受时间与氢溴酸右美沙芬比 较， 耐受时间延长， 具有良好的止咳作用。 实施例三十一： 柚皮素药物的祛痰药理学实验

From the naringenin sample and the positive control drug dextromethorphan hydrobromide, the tolerance time of naringenin in mice was significantly longer than that of the positive control drug. Dextromethorphan hydrobromide has a long tolerance time and is statistically significantly different. From the comparison between the naringenin sodium salt sample and the positive control drug dextromethorphan hydrobromide, the tolerance time of cough induced by irritated mice, the naringenin sodium salt has a significantly longer tolerance time in mice It is longer tolerated than the positive control drug dextromethorphan hydrobromide, which is statistically significantly different. This shows that naringenin and naringenin salt have a better tolerance to cough caused by irritated mice than dextromethorphan hydrobromide. Example 31: Expectorant Pharmacology Experiment of Naringenin

 1. Experimental animals: NIH mice, female, weighing 18.0-22.1g, clean grade standard, a total of 90 animals. The animals were weighed, numbered, and 80 healthy mice weighing 18.5-22.0 grams were selected. Sorted by weight, divided into eight groups by random grouping, 10 in each group. Negative control, positive control and naringenin 髙, medium, and low, and naringenin sodium high, medium, and low dose groups were set. All were administered orally at a dose of 10ml / kg body weight.

 2. Sample source and processing:

 1) Blank control group: saline, 0.9% NaCI.

 2) Positive control group: 0.2 g of Tankejing powder was dissolved in 10 ml of physiological saline to obtain the positive control phlegm solution with a concentration of 20mg / ml.

 3) Naringenin sample low-dose group: Precisely weigh 5mg of naringenin sample, make up to 10ml volumetric flask with physiological saline, dissolve by ultrasonic wave for 5 minutes, and shake to obtain. The concentration was 0.5 mg / ml.

4) Medium dose group of naringenin sample: Weigh 15mg of naringenin sample accurately, make up to a 10ml volumetric flask with normal saline, dissolve it with ultrasound for 5 minutes, and shake to obtain. The concentration was 1.5 mg / ml. 5) High-dose group of naringenin sample: Precisely weigh 45 mg of naringenin sample, make up to 10ml volumetric flask with physiological saline, dissolve by ultrasonic for 5 minutes, and shake to obtain. The concentration was 4.5 mg / ml.

 6) Low-dose group of naringenin sodium salt: Precisely weigh 5mg of naringenin sodium salt sample, make up to 10ml volumetric flask with physiological saline, dissolve by ultrasonic for 5 minutes, and shake to obtain. The concentration was 0.5 mg / ml.

 7) Medium dose group of naringenin sodium salt sample: Weigh 15mg of naringenin sodium salt sample accurately, make up to a 10ml volumetric flask with physiological saline, dissolve it with ultrasound for 5 minutes, and shake to obtain. The concentration was 1.5 mg / ml.

 8) High-dose group of naringenin sodium salt: accurately weigh 45mg of naringenin sodium salt sample, make up to a 10ml volumetric flask with physiological saline, dissolve it with ultrasound for 5 minutes, and shake to obtain. The concentration was 4.5 mg / ml.

 3. Experimental method:

 1) Drawing of standard phenol red curve: Weigh out a certain amount of phenol red with an analytical balance, dissolve it with 5% sodium bicarbonate, make up 12.5ng per 1 ml, and then serially dilute to phenol containing per ml Red 6,25ng, 3.125ng, 1.5625ng, 0.7813ng, 0.3906ng, 0.1953ng, 0.0977ng, 0.0488ng, OD value was measured by spectrophotometer. Using the phenol red concentration as the ordinate and the OD value as the abscissa, the regression equation was calculated based on the phenol red concentration and the OD value. The excretion of each phenol red was calculated according to the regression equation.

 2) Mice fast for 12 hours.

 3) Oral administration. In the order of the animal number, each mouse was stopped for 3 minutes after gavage, and then the other was given at an interval of 3 minutes. The total gavage time for each group of 10 mice was 30 minutes.

 4) 0.5 hours after intragastric administration of each rat, 0.2ml 5% phenol red physiological saline solution was injected intraperitoneally. In order, each mouse was injected intraperitoneally 3 minutes after phenol red, and then the other one was injected for 10 mice for a total of 30 minutes.

 5) 0.5 hours after intraperitoneal injection of each mouse, the mice were sacrificed in order to sacrifice the cervical spine, and the sacrifice interval was 3 minutes. After the animals were sacrificed, they were fixed on the operation board in the supine position, the skin in the middle of the neck was cut, the trachea was separated, and the trachea was supported with small forceps.

 6) Use a large syringe to absorb the saline solution to rinse the outer wall of the trachea, wash off the blood and the phenol red in the outer wall of the trachea, and filter the paper to absorb the dry cleaning solution.

 7) Cut the trachea before the branch of the trachea, then cut the trachea from the upper end of the other side of the thyroid cartilage (including the circular thyroid cartilage).

8) Put each trachea segment into a test tube containing 1.5ml of 5% NaHC0 ₃ solution in advance.

 9) Complete the tracheal separation and shearing work within 3 minutes. The second mouse was treated the same way. The method is as above.

 10) Put each test tube on an ultrasonic cleaner for 5 minutes to release the phenol red in the trachea section.

 11) The OD value of the solution in each test tube was measured at 546 nm of a 721 spectrophotometer.

 12) Leave the test tube containing the trachea section overnight, and re-measure the OD value after 24 hours.

 13) Calculate the content of phenol red according to the regression equation. Calculation formula: Y = 7.6266X_0.0554. X is the OD value and Y is the phenol red content.

 14) Calculate the corrected phenol red content based on the phenol red content and animal weight, and perform analysis of variance using SPSS 8.0 statistical software. Calibration Phenol Red Content = Phenol Red Content (ng) / Mouse Weight (kg)

 5. Experimental results:

 According to statistics, the phenol red excretion amount of each dose group is shown in Table 6 below. Table 6. The expectorant effect of naringenin and its salt (X ± S)

 Group

Dose animal tracheol corrected phenol red content ^a expectoration rate ^b

 P-value

Code Sample name (mg / kg) Red content (ng) (ng / kg) (%)

1 Saline 0 09385 ± 0.4148 467282 ± 22 · 6106 ― ―

2 Phlegm and cough net 200 1.3343 ± 0.7932 70.3811 ± 43.6970 P <0.05 150.62

3 Naringenin low-dose group 5 1.2757 ± 0 · 4020 68.2832 ± 23.6102 P <0.05 146.13

4 Naringenin medium-dose group 15 1.3523 ± 0.9303 74.7504 + 57.2020 P <0.05 159.97

5 Naringenin high-dose group 45 19257 ± 06713 102.7325 ± 41.0313 P <0.01 219.85 Naringenin sodium salt 69.4942+ 16.3724

 6 5 1.3029 ± 0.3069 ρ <0 · 05 148.72 Low dose group

 Naringenin sodium salt 758773 ± 392651

 7 15 1.4225 ± 0.7361 RP <0.05 162.38 Medium dose group

 Naringenin sodium salt 105.1057 ± 36.7945

 8 45 1.9705 ± 0.6898 P <0.01 224.93 High dose group

a _: Corrected phenol red content = phenol red content / animal weight

 b: expectoration rate = dosing group / blank control group X 100%

 Phenol red content calculation method (ng): OD value X7.6266- -0.0554 According to the experiment of increasing the bronchial secretion of mice by oral administration of naringenin sample and positive drug Tankejing, naringenin is low, medium and high Compared with the blank control group, the secretion of mouse bronchial secretion in each dose group of the dose group was significantly increased, and there were statistically significant differences. Compared with the positive control drug Tantanjing in the high-dose group, the bronchial secretion of mice was significantly increased, and the difference was statistically significant. The naringenin sodium salt sample and the positive drug phlegm and ketone were added to the bronchial secretion of mice by oral administration. It was found that the naringenin sodium salt was low, the medium and high dose groups were secreted by the mice Compared with the blank control group, there is a significant increase, and there is a statistically significant difference. Compared with the positive control drug Tankejing in the high-dose group, the bronchial secretion of the mouse increased significantly, which was statistically significantly different, and the effect was significantly higher than that of the positive drug Tankejing. This shows that naringenin and naringenin have good expectorant effects. Example 32: Toxicology test of naringin drug

 At a temperature of 24 ± C and a humidity of 65 ± 5%, 20 healthy clean-grade ΝΠΗ mice aged 7-8 weeks were selected, half male and half male, weighing 20-22 g. Feed and water were sterilized, and they were maintained under normal feed conditions before and during the observation period.

 Naringin was dissolved in 0.5% Tween80 at a concentration of 300 mg / ml, and the liquid was orally administered to mice at a dose of

0.2ml / 20g mouse body weight. Observe for 1, 4, 8, and 12 hours after administration, and then every 12 hours thereafter. Observe death, record changes in mouse body weight and other symptoms every day. On day 10, the mice were sacrificed by neck dissection, and each organ was taken for pathological examination.

 On the 10th day, all mice survived, and no toxic reaction was observed at the 3.0 g / kg dose of naringin. This dose is equivalent to 230 times the normal dose. The pathological examination of the mice's organs was normal, no lesions were found, and no weight loss was observed in the mice within 10 days. Therefore, it is shown that the naringin drug of the present invention shows no toxicity when administered orally to animals. Example 33: Toxicology Experiment of Naringin Sodium Drug

 At a temperature of 25 ± C and a humidity of 75 ± 5%, 20 healthy clean-grade MH mice aged 7-8 weeks were selected, half male and half female, weighing 18.5-20.5 g. Feed and water were disinfected, and they were kept under normal feed conditions before and during the observation period.

 The naringin sodium salt was dissolved in physiological saline at a concentration of 300 mg / ml, and the liquid was orally administered to mice at a dose of

0.2ml / 20g mouse body weight. Observe for 1, 4, 8, and 12 hours after administration, and then every 12 hours thereafter. Observe death, record changes in mouse body weight and other symptoms every day. On day 10, the mice were sacrificed by neck dissection, and each organ was taken for pathological examination.

 On the 10th day, all mice survived, and no toxic reaction was observed at the 3.0 g / kg dose of naringin sodium salt. This dose is equivalent to 230 times the normal human dose. The pathological examination of the mice's organs was normal, no lesions were found, and no weight loss was observed in the mice within 10 days. Therefore, it is shown that the naringin sodium salt drug of the present invention has no toxicity when administered orally to animals. Example 34: Toxicology Experiment of Naringenin Drugs

 At a temperature of 28 ° C and a humidity of 70 ± 5%, 20 healthy clean Kunming mice aged 7-8 weeks were selected, half male and half male, weighing 19 to 21 g. Feed and water were disinfected, and they were kept under normal feed conditions before and during the observation period.

Naringenin 'was dissolved in 0.5% Tween80 at a concentration of 300 mg / ml, and the liquid was orally administered to mice at a dose of 0.4 ml / 20 g of mouse body weight. Observe for 1, 4, 8, and 12 hours after dosing, and then every 12 hours thereafter. Observe death, record changes in mouse body weight and other symptoms every day. On day 10, the mice were sacrificed by neck dissection, and each organ was taken for pathological examination. On the 10th day, all mice survived, and no toxic reaction was observed with naringenin at a dose of 6.0 g / kg. This dose is equivalent to 460 times the normal human dose. The pathological examination of the mice's organs was normal, no lesions were found, and no weight loss was observed in the mice within 10 days. Therefore, it was demonstrated that the naringenin drug of the present invention showed no toxicity when administered orally to animals. Example 35: Toxicology test of naringenin sodium drug

 Under the condition of 27 ± C and 60 ± 5% humidity, 20 healthy clean Kunming mice were selected from 7-8 weeks old, half male and half male, weighing 18.2-22.5g. Feed and water were sterilized. Before the test and the observation period of the test, they were raised according to normal feed conditions.

 The naringenin sodium salt was dissolved in physiological saline at a concentration of 300 mg / ml, and the liquid was orally administered to mice at a dose of 0.4 ml / 20 g of the body weight of the mice. Observe for 1, 4, 8, and 12 hours after administration, and then every 12 hours thereafter. Observe death, record changes in mouse body weight and other symptoms every day. On day 10, the mice were sacrificed by neck dissection, and each organ was taken for pathological examination.

 On the 10th day, all mice survived, and there was no toxic reaction with naringenin sodium salt at a dose of 6.0 g / kg. This dose is equivalent to 460 times the normal human dose. The pathological examination of the mice's organs was normal, no lesions were found, and no weight loss was observed in the mice within 10 days. Therefore, it is shown that the naringenin sodium salt drug of the present invention has no toxicity when administered orally to animals. Example 36: Naringin Capsule Preparation

 Prepare gelatin capsules with the following ingredients:

 Naringin 100g

 Dry starch 40g

 Micro powder silica gel 10g

Mix the excipients and naringin evenly and put them into transparent capsules to obtain. Filling: 150mg / capsule. Example 37: Naringin tablets

 Tablets are prepared in the following proportions:

 Naringin 750g

 Starch 722.5g

 Starch slurry (14%) 25.0g

 Magnesium Stearate 2.5g

 Total 1500g

 Mix naringin with starch uniformly, add starch slurry and continue to stir to make a soft material, granulate with a 10 mesh nylon sieve, 80-90Ό ventilate and dry, add magnesium stearate to the dry granules, adjust the granules through a 12 mesh sieve, mix the hooks , Compressed into tablets. A total of 5000 tablets were obtained, each weighing about 0.3g. Example 38: Naringenin capsules

 Prepare capsules with the following ingredients:

 Naringenin 60g

 Dry starch 35g

 Micro powder silicone 5g

Mix the excipients and naringenin evenly and fill them in gelatin capsules to obtain. Filling: 100mg / capsule. Example 39: Naringenin tablets

 Tablets are prepared in the following proportions:

 Naringenin 500g

 Starch 472.5g

 Starch Paddle (14%) 25.0g

 Magnesium Stearate 2.5g

1000g in total Mix naringenin and starch, add starch firewood and continue stirring to make a soft material, granulate with a 10 mesh nylon sieve, 80-9CTC ventilate and dry, add magnesium stearate to the dry granules, adjust the granules through a 12 mesh sieve, mix Mix well and compress into tablets. A total of 10,000 tablets were obtained, each weighing about 0.1 g. Example 40: Naringenin inhalant

 Prepare inhalants at the following distribution ratios:

 Naringenin 100g

 500g lactose

 Polosham ig

 Micro powder silica gel 10g

 L-leucine 0.5g

 PEG400 (50%) aqueous solution 300g

 Total 911.5g

 The naringenin, lactose, poloxamer, and L-leucine were dissolved in an aqueous solution of PEG400, and then spray-dried. The resulting spray-dried powder was added to micronized silica gel, mixed uniformly, and dispensed into a capsule-type dry powder inhalation device. Example 41: Naringin Sodium Injection

 Take 100 g of naringin sodium salt, add 10,000 ml of water for injection to dissolve the sample, adjust the pH value to 7 ~ 8 with hydrochloric acid, and finally filter through a G3 vertical melting funnel, pot, and sterilize with 10CTC steam for 30 minutes to obtain naringin Sodium glycoside injection. Example 42: Naringenin Sodium Injection

 Take 100g of naringenin sodium salt, add 10 liters of water for injection to dissolve the sample, adjust the pH value to 7 ~ 8 with hydrochloric acid, finally filter through a G3 vertical melting funnel, pot, and sterilize with 10CTC steam for 30 minutes to obtain naringin Sodium injection. Example 43: Naringin syrup

 Take 100g of naringin, add 650g of sucrose, stir well, add 800ml of distilled water, heat and boil to dissolve, filter, cool, add preservatives, and dilute to 1000ml with distilled water to obtain naringin syrup. Example 44: Naringenin

 Take 100g of naringenin, add 500g of sucrose, stir well, add 800 ml of steamed simmered water, heat and boil to dissolve, filter, cool, add preservatives, and dilute to 1000 ml with distilled water to obtain naringenin syrup . Embodiment 45: Naringenin sodium salt powder injection

 Take 100 g of naringenin sodium salt, dissolve it in 10,000 ml of distilled water, sterilize it at 121 ° C for 15 minutes, take it out, fill it in a 1 ml ampoule aseptically, freeze freeze dry it, and seal it under sterile conditions. Obtain naringenin sodium powder. Example 46: Naringin Sodium Powder Injection ''

 Take 100g of naringin sodium salt, dissolve it by adding 10 liters of distilled water, sterilize it at 12TC for 15 minutes, take it out, aseptically fill it in a 1 ml ampoule, freeze freeze dry, and seal it under aseptic conditions to obtain pomelo Hesperidin sodium powder.

Claims

Claim 1. Application of naringenin, naringin and its salt in preparing a pharmaceutical composition for relieving cough and expectorant. 2. A pharmaceutical composition for relieving cough and expectorant, characterized in that it comprises an effective therapeutic dose of naringenin, naringin and its salts as active ingredients, and a pharmaceutically acceptable carrier. 3. The pharmaceutical composition according to claim 2, characterized in that: the effective therapeutic dose is 0.1 to 500 mg / kg body weight / day. 4. The pharmaceutical composition according to claim 2, characterized in that: the pharmaceutical composition is formulated into a dosage form selected from the group consisting of a tablet, a sugar agent, a capsule, a powder injection, an injection, and an inhaler. 5. The pharmaceutical composition according to claim 2, characterized in that the pharmaceutical composition is used for oral administration, injection administration, or pulmonary inhalation administration. 6. The pharmaceutical composition according to claim 2, wherein the naringin and its salt are synthesized by a chemical method. 7. The pharmaceutical composition according to claim 2, wherein: the naringin is a naringin monomer obtained by extracting from a naringin-containing plant raw material. 8. The pharmaceutical composition according to claim 7, wherein the method for extracting naringin comprises the steps of: pulverizing a naringin-containing plant raw material, extracting it with water for one to three times, filtering, and combining the filtrates The filtrate is concentrated into an extract, which is loaded onto the column or directly after the alcohol is precipitated, and the column is separated, and the organic solvent is eluted; the solvent is recovered from the eluent to obtain the crude naringin; and then recrystallized one to ten times to obtain Naringin monomer. The pharmaceutical composition according to claim 2, wherein the naringenin is a naringenin monomer obtained by hydrolyzing naringenin. 10. The pharmaceutical composition according to claim 9, wherein the extraction method of naringenin comprises the following steps: dissolving naringin in water, stirring uniformly, adding acid or alkali or enzymatic hydrolysis, and precipitating and filtering After that, the crude naringenin is obtained; and then it is passed through the organic solvent one to ten times. Recrystallization to obtain naringenin monomer. 11. The pharmaceutical composition according to claim 10, wherein the enzyme is a glucuronidase or other enzyme capable of cleaving a six-carbon sugar. 12. The pharmaceutical composition according to claim 2, wherein the naringenin is a naringenin monomer extracted from a naringenin-containing plant raw material. 13. The pharmaceutical composition according to claim 12, wherein the method for extracting naringenin comprises the steps of: pulverizing a naringenin-containing plant raw material, extracting it with an organic solvent one to three times, and filtering, The filtrates were combined; the filtrates were concentrated and precipitated to obtain a crude naringenin; and the organic solvents were recrystallized one to ten times to obtain a naringenin monomer. . 14. The pharmaceutical composition according to claim 2, wherein: the naringenin is a naringenin monomer obtained by directly hydrolyzing from a naringin-containing plant raw material. 15. The pharmaceutical composition according to claim 14, wherein the naringenin extraction method comprises the steps of: pulverizing a naringin-containing plant raw material, extracting it with water or an organic solvent one to three times, and filtering, The filtrates were combined; the filtrates were concentrated to extracts, the extracts were dissolved in water or organic solvents, added with acid or alkali or enzymatic hydrolysis, and filtered to obtain the crude naringenin; and the naringenin monomers were obtained after one to ten recrystallizations. 16. The pharmaceutical composition according to claim 15, wherein the enzyme is a glucuronidase or other enzyme capable of cleaving a six-carbon sugar. 17. The pharmaceutical composition according to claim 2, wherein: the naringenin salt is a metal salt formed by the reaction of naringenin and a corresponding basic substance, and the molecular formula is: C ₁₅ H ( ₁₂ . _n p ₅ Y _n , n = 1-3, Y is a metal ion. 18. The pharmaceutical composition according to claim 17, characterized in that the naringenin salt is prepared by the following method: Under the condition of 0 ~ 10CTC, naringenin reacts with the corresponding alkaline substance to generate the corresponding A metal salt of naringenin; during the reaction, the molar ratio of naringenin to the alkaline substance is 1: 1 to 1: 3. 19. The pharmaceutical composition according to claim 17 or 18, wherein the basic substance is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate. One, the naringenin salt obtained by the reaction is the sodium or potassium salt of naringenin, and its molecular formula is: C ₁₅ H _(12-n) 0 ₅ Y _n , η = 1-3, γ is κ ion or Na ion. 20. The pharmaceutical composition according to claim 2, wherein: the naringin salt is a metal salt formed by the reaction of naringenin and a corresponding basic substance, and the molecular formula is: C ₂₇ H ( ₃₂ . _n ) 0 ₁₄ Y _n , n = 1-2, Y is a metal ion. 21. The pharmaceutical composition according to claim 20, wherein the naringin salt is prepared by the following method: Under the condition of 0 ~ 40 ° C, naringin reacts with the corresponding basic substance to form The metal salt of naringin; during the reaction, the molar ratio of naringin to basic substance is 1: 1 to 1: 2. 22. The pharmaceutical composition according to claim 20 or 21, wherein the basic substance is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate. One, the naringenin salt obtained by the reaction is the sodium or potassium salt of naringin, and its molecular formula is: C ₂₇ H ( _32-n ) 0 ₁₄ Y _n , n = 1-2, Y is K ion or Na ion. 23. A method for treating cough and expectorant, characterized in that it comprises administering the pharmaceutical composition according to claim 2 to a mammal. 24. The method of claim 23, wherein the mammal is a human.