CN1555793A - Naringenin and its salts used in the preparation of antitussive and expectorant drugs - Google Patents

Abstract

The invention relates to the application of naringenin (Naringenin) and its salt in the preparation of medicine for relieving cough and reducing phlegm. The naringenin used can be obtained by hydrolysis of crude naringin (Naringin) or monomers, or extracted from various medicinal materials containing naringin, or synthesized by other chemical methods; the naringenin salt can be Obtained by the reaction of naringenin with the corresponding base. The medicine with naringenin or/and naringenin salt as an active ingredient has a good effect of relieving cough and reducing phlegm, and has a remarkable curative effect on various coughs and asthma caused by acute and chronic bronchitis and colds, without toxic and side effects.

Description

Naringenin and its salts used in the preparation of antitussive and expectorant drugs

technical field

The present invention relates to the use of naringenin (Naringenin) and its salt for preparing medicine for relieving cough and reducing phlegm.

Background technique

Cough, sputum, and wheezing are common symptoms of respiratory diseases. They are especially common in daily life caused by acute and chronic bronchitis, colds, etc. Clinically, cough, sputum, wheezing and recurrent attacks are characterized by infection or Acute and chronic non-specific inflammation of the trachea, bronchial mucosa and surrounding tissues caused by non-infectious factors. It belongs to the category of cough, phlegm retention, and asthma in traditional Chinese medicine, and is a common and frequently-occurring disease. Especially middle-aged and elderly people are prone to bronchitis symptoms such as cough and expectoration due to the influence of environmental factors and living habits. They are not well treated and controlled in the early stage of the disease, and gradually develop into diseases.

For the use of traditional Chinese medicine to relieve cough and reduce phlegm, people have carried out a lot of efforts, such as Juhong Wan and Keke Tablets, which have entered the market. But in terms of its characteristics, the currently marketed drugs also have the disadvantage of large doses and inconvenience. Therefore it is necessary to continue to develop new drugs for relieving cough and resolving phlegm that are safe and reliable in efficacy. Naringenin (Naringenin), the chemical name is 5,7,4'-trihydroxydihydroflavone, is a flavonoid compound naringin (naringin) aglycone, widely present in cherry blossom buds, plum buds and Citrus aurantium , In plant tissues such as citrus, it is a kind of flavonoids, which has antibacterial, anti-inflammatory, anti-cancer, antispasmodic and choleretic effects, treats cardiovascular diseases, and lowers cholesterol. However, there is no report on the application of naringenin in relieving cough and reducing phlegm.

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

The structural formula of naringenin:

Naringenin

Contents of the invention

The purpose of the present invention is to provide the application of naringenin (naringenin) and its salt in the preparation of medicine for relieving cough and reducing phlegm.

The naringenin salt in the present invention refers to the metal salt generated by the reaction of naringenin with the corresponding alkali (usually sodium hydroxide or potassium hydroxide) (ie naringenin molecule 5, 7 or/and 4' The H on the hydroxyl group is replaced by an alkali metal ion, usually naringenin sodium or potassium salt). The molecular formula of naringenin salt is: C ₁₅ H _n O ₅ Y _m , n=9-11, m=1-3, Y is a metal ion (usually sodium or potassium ion).

The present invention proves through pharmacological and pharmacodynamic experiments that naringenin or/and naringenin salt has a good effect of relieving cough and reducing phlegm, and has obvious curative effect on diseases such as cough and phlegm caused by acute and chronic bronchitis and colds.

We used naringenin and naringenin salts to carry out antitussive experiments in small rats. The results showed that: compared with the blank control group, the tolerance time of naringenin and naringenin salt to the cough caused by stimulating mice was significantly prolonged, and there was a statistically significant difference; Compared with dextromethorphan tablets, the tolerance time was prolonged, and the curative effect was significantly better than that of the positive control drug dextromethorphan hydrobromide tablets.

We use naringenin and naringenin salts to conduct phlegm-reducing experiments on experimental animals, mice. The results showed that: naringenin and naringenin 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 Tankejing, it has a significant increase in the bronchial secretion of mice, and there is a statistically significant difference, and the curative effect is more significant than that of the positive drug Tankejing. It shows that naringenin and naringenin salt have the effect of resolving phlegm.

Experiments of the present invention prove that naringenin or/and naringenin salt not only has a good effect of relieving cough and reducing phlegm, but also shows little toxicity in mouse animal experiments. Animal experiments show that when 300mg/kg dose of naringenin or naringenin salt is orally administered to animals, no toxic reaction is seen in animals, which is equivalent to a dose of 4-6g naringenin or naringenin salt/ kg body weight.

In summary, it shows that naringenin or/and naringenin salt has a good effect of relieving cough and reducing phlegm, without toxic and side effects, and can be very good for treating cough and phlegm caused by acute and chronic bronchitis and colds in clinical practice. many. Therefore, it can be used to prepare cough-relieving and phlegm-reducing medicines.

The medicament for relieving cough and reducing phlegm (hereinafter referred to as naringenin drug) prepared by using naringenin or/and naringenin salt in the present invention can contain 0.1-100%wt.naringenin or/ and naringenin salt. Said naringenin medicine can simply be made up of naringenin or/and naringenin salt monomer, or be made up of naringenin or/and naringenin salt as active ingredient and other active ingredient or/and conventional pharmaceutical Excipient composition. When naringenin and naringenin salt are used together, there can be any ratio between the two.

The above-mentioned naringenin drug can be prepared into required pharmaceutical preparations in different dosage forms by conventional methods by selecting conventional auxiliary materials suitable for corresponding dosage forms or without adding auxiliary materials. The added auxiliary materials can be solid, semi-solid or liquid substances, and serve as carriers, excipients or media for naringenin or/and naringenin salt. Therefore, said naringenin and its salt pharmaceutical preparations can be tablets, powders, sachets, sweet liquors, suspensions, emulsions, solutions, syrups, aerosols, inhalants, soft or hard Capsules, sterile injections and other dosage forms.

The capsule of the above-mentioned naringenin medicine, its content contains naringenin or/and naringenin salt of 0.5-100%wt.; usually can be made up of not less than naringenin or/and and naringenin salt and other active ingredients or/and various conventional auxiliary materials. The tablet of said naringenin medicine contains not less than 0.1% wt. naringenin or/and naringenin salt in its composition; it can be composed of not less than 0.1% wt. The naringenin salt is composed of other active ingredients or/and various conventional auxiliary materials. The inhalation of said naringenin medicine, its content contains naringenin or/and naringenin salt of 0.1-100%wt.; Usually can be made up of not less than naringenin or/and naringenin of 0.1%wt. The naringenin salt is composed of other active ingredients or/and various conventional auxiliary materials.

The naringenin drug described in the present invention has a good effect of relieving cough and reducing phlegm at 0.1-700mg naringenin or/and naringenin salt/kg body weight/day, and the preferred daily dose is about 1-100mg naringenin or / and naringenin salt/kg body weight/day.

The naringenin medicine of the present invention has a good effect of relieving cough and resolving phlegm. It can effectively treat cough and phlegm caused by acute and chronic bronchitis and colds. And it has the characteristics of stable quality, small dosage, rapid curative effect and safety.

The naringenin mentioned in the present invention can be crude naringenin or naringenin monomer (pure product) prepared by one of the following methods:

Method 1: Obtain from crude naringin (naringin) or monomer by hydrolysis. The method of hydrolysis may be acid hydrolysis, alkaline hydrolysis or enzymatic hydrolysis. The specific method can be carried out according to the following steps: add water to dissolve the crude naringin or monomer, stir evenly, add acid, alkali or enzyme, and mix evenly; the mixed solution is hydrolyzed at room temperature/heating; after the hydrolysis is completed, precipitate and filter to obtain crude naringenin ; Then recrystallized by organic solvent or water several times to get naringenin monomer.

In the above-mentioned method one, the amount of water (volume) added by hydrolysis can be 1 to 1000 times of the crude product of naringin or the monomer (weight), and the amount of water added is not limited; the acid in the acid hydrolysis can be hydrochloric acid, sulfuric acid, phosphoric acid or For various acids such as nitric acid, the amount of acid can be added to make the solution PH≤7, the temperature of acid hydrolysis can be from room temperature to 100°C, and the time of hydrolysis can be 0.1 to 170 hours; the alkali in alkali hydrolysis can be Sodium hydroxide, potassium hydroxide, calcium hydroxide and other inorganic alkalis, the amount of alkali can be added to make the solution pH ≥ 7, the temperature of alkali hydrolysis can be from room temperature to 100 °C, and the hydrolysis time can be 0.1 ~170 hours; the enzyme in the enzymatic hydrolysis can be various enzymes that break down six-carbon sugars such as glucuronidase, and the dosage of the enzyme can be such that the concentration of the enzyme in the solution is 0.1 micromol/liter to 0.1 mole/ml after adding the enzyme , the temperature of enzymatic hydrolysis can be from room temperature to 70°C, and the time of hydrolysis can be from 0.1 to 120 hours. The acid, alkali or enzymatic hydrolysis time is not limited.

In the above-mentioned method one, the used naringin crude product or monomer can be obtained from various medicinal materials containing naringin (for example: Fructus Fructus Fructus Fructus Fructus Aurantii, Citrus Fructus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus Citrus Fructus, orange, lemon , grapefruit, tangerine, orange, pomelo, etc.); can also be synthesized by chemical methods.

Method 2: extract crude naringenin or monomeric naringenin (pure product ). The extraction method can be carried out according to the following steps: crush the medicinal materials, extract one or more times with an organic solvent, filter, and combine the filtrate; Second recrystallization, naringenin monomer was obtained.

In the above-mentioned method two, the organic solvent used for organic solvent extraction can be various commonly used organic solvents such as ethanol, methyl alcohol, chloroform, ethyl acetate, acetone or sherwood oil, can adopt the organic solvent of various concentration (comprising that concentration is 100%) ; Organic solvent extraction can be carried out at room temperature to 100°C or/and ultrasonic conditions. The number of extractions is generally one to three times, and the number of times can be increased without limitation; Each extraction time is generally 0.1-144 hours, and the time extension is not limited; extraction under heating or/and ultrasonic conditions can shorten the extraction time.

In above-mentioned method one and method two, the used organic solvent of recrystallization can adopt various commonly used organic solvents such as ethanol, methanol, chloroform, ethyl acetate, acetone or sherwood oil, and the number of times of recrystallization can be one to ten times, and the number of times increases unlimited.

Using the above-mentioned method one and method two of the present invention, naringenin (pure product) is obtained from the hydrolysis of naringin and extracted from different naringenin/naringin medicinal materials to obtain naringenin (pure product). The yields are all above 85%, and the extraction yield is high. The obtained naringenin monomer (pure product) is determined by a high-performance liquid chromatography of "Pharmacopoeia of the People's Republic of China" 2000 edition, and is quantified by the external standard method with the naringenin reference substance provided by the China Pharmaceutical and Biological Products Inspection Institute , to obtain the content of naringenin monomer in the extracted naringenin sample. The results showed that the content of naringenin monomer in the sample was higher than 95%. It shows that the process of the present invention is simple and feasible.

The above-mentioned naringenin preparation method of the present invention has the characteristics of simple extraction process, high extraction rate, and high purity of extracted naringenin monomer.

Method 3: synthesized by other chemical methods.

Detailed ways

Below in conjunction with embodiment the present invention will be further described.

The ratios of solids and solids, liquids and liquids, and liquids and solids in the material dosage proportions and component contents involved in each embodiment are represented by wt/wt (weight ratio), v/v (volume ratio), wt/v (weight/volume ratio) calculations unless otherwise stated. The naringin monomer and crude naringin used in Examples 1-4 were prepared according to the method disclosed in Chinese patent application CN1431216A. The naringenin used in Examples 15-20 is the naringenin monomer obtained by the method described in Examples 1-6 respectively, and the purity is above 95%.

Example 1:

Naringin monomer (purity 97.3%), add 10 times of water, mix evenly, add concentrated hydrochloric acid to make the solution PH = 1, mix evenly; heat and hydrolyze at 100°C for 2 hours; after hydrolysis, filter to obtain crude naringenin ; After five times of recrystallization with absolute ethanol, naringenin monomer was obtained. The yield was 91.4%.

Example 2:

For crude naringin (purity 98.2%), add 20 times of water, mix evenly, add concentrated sulfuric acid to make the solution pH = 0, mix evenly; heat and hydrolyze at 80°C for 3 hours; after hydrolysis, filter to obtain crude naringenin; After six times of recrystallization with ethyl acetate, naringenin monomer was obtained. The yield was 89.3%.

Example 3:

Crude naringin (purity 98.5%), add 50 times of water, mix evenly, add sodium hydroxide solution to make the solution PH = 13, mix evenly; heat and hydrolyze at 80°C for 1.5 hours; after hydrolysis, filter to obtain naringenin Crude product; after three times of recrystallization with acetone, naringenin monomer was obtained. The yield was 86.9%.

Example 4:

Naringin monomer (purity 96.7%), add 35 times of water, mix evenly, add glucuronidase to make the concentration of enzyme in the sample is 0.1 mmol/L, mix evenly; heat and hydrolyze in 37 ℃ water bath for 8 hours; hydrolysis ends After filtering, the crude product of naringenin was obtained; after four times of recrystallization with absolute ethanol, the monomer of naringenin was obtained. The yield was 94.8%.

Example 5:

Cherry blossom buds, add absolute ethanol to submerge the medicinal materials, extract three times, the extraction temperature is 50°C, one hour each time, and filter; the filtrate is concentrated into an extract, and precipitation occurs, filtered to obtain crude naringenin; the precipitation is obtained with ethyl acetate The ester crystallized three times to obtain naringenin monomer. The yield was 90.7%.

Embodiment 6:

Plum flower buds, add acetone to submerge medicinal materials, soak at room temperature, extract three times, 48 hours each time, filter, and combine the filtrate; After five times of recrystallization, naringenin monomer was obtained. The yield was 87.4%.

Embodiment 7:

Cut the medicinal material of Citrus aurantium into decoction pieces, extract twice with water at 100°C for 3 hours each time, filter, and combine the filtrate; Crystallize six times to get naringenin monomer. The yield was 85.3%.

Embodiment 8:

The grapefruit medicinal material is not crushed, extracted 4 times with ethyl acetate by ultrasound, 1 hour each time, filtered, and the filtrates are combined; concentrated to obtain the extract, and the extract is left overnight to produce precipitation, separate the precipitate, dry the precipitate, and obtain pomelo peel The crude product of naringenin; the precipitate was recrystallized twice with acetone to obtain naringenin monomer. The yield was 86.1%.

Embodiment 9:

Crush the medicinal material of Citrus aurantium, pass through a 20-mesh sieve, extract three times with water at room temperature, and filter for two days each time, and combine the filtrate; concentrate the filtrate, add concentrated hydrochloric acid to make the liquid pH = 3, heat and hydrolyze at 90°C, and when the hydrolysis is completed, filter, The precipitate was obtained, and the precipitate was dried to obtain crude naringenin; the precipitate was recrystallized four times with ethyl acetate to obtain naringenin monomer. The yield was 90.7%.

Example 10:

Grapefruit herbs are cut into decoction pieces, add 100°C water to submerge the herbs, extract twice, each time for 2 hours, filter, and combine the filtrate; the filtrate is concentrated, add sodium hydroxide to the solution pH = 11, heat and hydrolyze at 70°C, the hydrolysis is completed, Filtrate to obtain a precipitate, dry the precipitate to obtain crude naringenin; recrystallize the precipitate three times with acetone to obtain naringenin monomer. The yield was 89.2%.

Example 11:

The tangerine medicinal material is not crushed, extracted with acetone 4 times, 24 hours each time, filtered, and the filtrates are combined; concentrated to obtain an extract, which is dissolved by adding 30% ethanol, and concentrated phosphoric acid is added to make the liquid PH = 2, heated and hydrolyzed at 95°C After the hydrolysis is completed, filter to obtain a precipitate, and dry the precipitate to obtain crude naringenin; the precipitate is recrystallized five times with chloroform to obtain naringenin monomer. The yield was 87.7%.

Example 12:

Cut the medicinal material of Fructus Fructus Fructus into decoction pieces, add 100°C water to submerge the medicinal material, add concentrated hydrochloric acid to make the solution PH=2.5, extract three times, each time for 1.5 hours, filter, and combine the filtrate; the filtrate is concentrated to obtain an extract, and the extract is left overnight to obtain Precipitate; separate the precipitate, dissolve it with acetone, filter, discard the precipitate, recover acetone from the filtrate, obtain the precipitate, dry the precipitate, and obtain crude naringenin; recrystallize the precipitate three times with chloroform to obtain naringenin monomer. The yield was 90.4%.

Example 13:

Shatian pomelo herbs are crushed, add 12 times of 100°C water to submerge the herbs, add potassium hydroxide to make the solution PH = 14, extract twice, each time for 45 hours, filter, and combine the filtrates; concentrate the filtrates to obtain extracts, and store the extracts overnight , to obtain a precipitate; separate the precipitate, dissolve it with absolute ethanol, filter, discard the precipitate, recover ethanol from the filtrate, obtain the precipitate, dry the precipitate, and obtain crude naringenin; recrystallize the precipitate four times with acetone to obtain naringenin monomer. The yield was 88.3%.

Example 14: Purity Determination Experiment of Naringenin Monomer

Take an appropriate amount of naringenin monomer prepared by the above extraction method, dissolve it in methanol and set the volume in a volumetric flask, filter it with a microporous membrane (0.45um), inject it into a high-performance liquid chromatograph, and test it with Chinese pharmaceutical and biological products The provided naringenin reference substance is quantified by the external standard method to obtain the content of naringenin monomer in the extracted naringenin sample. The results are shown in Table 1. Samples 1-11 in Table 1 are the naringenin monomers prepared in Examples 1-11, respectively.

Chromatographic conditions: Agilent1100 high performance liquid chromatography (autosampler, vacuum degasser, quaternary pump, column thermostat, diode array detector); chromatographic column: MARKER ODS column (5um, 4.0×250mm); mobile phase : methanol-water (50:50); detection wavelength: 288nm; column temperature: 30°C; flow rate: 1ml/min; injection volume: 5μl.

Table 1: Sample content determination test results

  Sample Concentration

                                                                             

sample number

Sample volume (μL) peak area

(mg/ml)

Degree (mg/ml) (%)

Naringenin

0.500 5 3145.74885 0.500 100

Reference substance

Sample 1 0.462 5 2850.04845 0.453 98.1

Sample 2 0.472 5 2925.54643 0.465 98.5

Sample 3 0.521 5 3189.78933 0.507 97.4

Sample 4 0.483 5 2925.54649 0.465 96.3

Sample 5 0.512 5 3189.78934 0.507 99.0

Sample 6 0.502 5 3019.91890 0.480 95.6

Sample 7 0.439 5 2661.30353 0.423 96.3

Sample 8 0.494 5 3032.50189 0.482 97.5

Sample 9 0.508 5 3095.41687 0.492 96.9

Sample 10 0.517 5 3196.08083 0.508 98.3

Sample 11 0.486 5 2988.46141 0.475 97.7

As can be seen from the results in Table 1, the content of naringenin monomers in the extracted samples is all higher than 95%, indicating that the naringenin samples obtained by the extraction method of the present invention have high purity.

It can be known from the above-mentioned Examples 1 to 14 that the preparation method of naringenin of the present invention can effectively obtain naringenin monomer by hydrolyzing the crude product/monomer of naringenin into naringenin, and can effectively obtain naringenin-containing The naringenin monomer is extracted from the naringin medicinal material, and the obtained naringenin sample has high purity, and the content is all higher than 95%. The process is simple and feasible.

Example 15: Antitussive pharmacological experiment of naringenin drug

1. Experimental animals: NIH mice, male, weighing 18.2-21.7g, normal-grade standard, 130 in total. Animals were first weighed, numbered, and a total of 120 healthy mice with a body weight of 18.5-21.0 g were selected. Sorted according to body weight, divided into eight groups by random grouping method, 15 in each group. A negative control group, a positive control group, a low-dose group of naringenin drug samples, a middle-dose group of naringenin drug samples, a high-dose group of naringenin drug samples, a low-dose group of naringenin sodium salt drug samples, and a naringenin drug sample low-dose group were set up. The middle dose group of the sodium salt drug sample and the high dose group of the naringenin sodium salt drug sample.

2. Sample source and processing:

1) Blank control group: physiological saline, NaCl content 0.9%.

2) Positive control group: two tablets of methorphan were dissolved in 20 ml of normal saline to obtain a positive control methorphan solution, the concentration of methorphan was 1.5 mg/ml.

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

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

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

6) Low-dose group of naringenin sodium salt samples: take an appropriate amount of naringenin sodium salt, dilute it in a volumetric flask with normal saline, and the concentration of naringenin sodium salt is 0.5 mg/ml.

7) Medium dose group of naringenin sodium salt sample: 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 1.5 mg/ml.

8) High-dose group of naringenin sodium salt samples: 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 4.5 mg/ml.

3. Experimental method: (concentrated ammonia water spray method)

One hour after gavage, the mice began to receive the spray. The concentrated ammonia water mist was sprayed for a certain period of time. After the spraying was over, the mice were immediately taken out to observe whether there was a cough reaction. Observe the number of coughs within one minute. If there are more than 3 typical coughing movements (abdominal muscle contraction or chest shrinkage, while opening the mouth wide, sometimes coughing) within 1 minute, it is counted as "cough". Otherwise count as "no cough".

4. Experimental process:

The nebulization time (EDT ₅₀ ) that caused half of the mice to cough was calculated by the sequential method (up and down method). Calculate the R value, if the R value is greater than 130%, it means that the medicine has antitussive effect. If the R value is greater than 150%, it indicates a significant antitussive effect. Calculated as follows:

EDT ₅₀ =log ⁻¹ c/n (wherein n is the number of animals, c is the sum of the rx values, r is the number of animals in each dose group, and x is the logarithm of the dose (i.e. spray time).)

5 Experimental results:

After statistics, the half cough time and cough-relieving effect of each sample group are shown in Table 1.

                                                    

Group

Administration dose Sample concentration EDT ₅₀ R value Antitussive

Code Name Sample Name (ml/20g) (mg/ml) (seconds) (%) Effect

1 Normal saline group 0.2 0 38.9 - -

2 methorphan group 0.2 1.5 53.70 138.05 effective

3 Naringenin sample low dose group 0.2 0.5 51.29 131.85 effective

4 Naringenin sample middle dose group 0.2 1.5 66.07 169.85 markedly effective

5 naringenin sample high dose group 0.2 4.5 66.61 171.23 markedly effective

6 Naringenin sodium salt sample low dose group 0.2 0.5 55.35 142.3 effective

7 Naringenin sodium salt sample medium dose group 0.2 1.5 69.48 178.6 markedly effective

8 Naringenin sodium salt sample high dose group 0.2 4.5 73.56 189.1 markedly effective

From the comparison of the tolerance time of naringenin samples and the positive control drug methorphan, oral administration to stimulate mice to cough, naringenin has a significant prolongation of the tolerance time of mice, which is higher than that of the positive control drug methorphan. There is a statistically significant difference over a long period of time. From the naringenin sodium salt sample and the positive control drug dextromethorphan, the tolerance time of oral administration to stimulate the mice to cough is compared. The tolerance time of dextromethorphan was longer and the difference was statistically significant. It shows that the tolerance time of naringenin and/or naringenin salt to the cough caused by stimulating mice is longer than that of methorphan, and the curative effect is remarkable. It has a good effect of relieving cough and reducing phlegm, and has obvious curative effect on diseases such as cough and phlegm caused by acute and chronic bronchitis and colds.

Example 16: Phlegm-reducing pharmacological experiment of naringenin drug

1. Experimental animals: NIH mice, female, weighing 18.0-22.1g, clean standard, certificate number: Yuejian Zhengzi No. 2002A022, provided by the Experimental Animal Center of First Military Medical University on October 12, 2003. A total of 90 pieces. Animals were first weighed, numbered, and a total of 80 healthy mice with a body weight of 18.5-22.0 grams were selected. Sorted according to body weight, divided into eight groups with random block grouping method, 10 in each group. Set up negative control, positive control and naringenin high, medium and low, and naringenin sodium salt high, medium and low eight dosage groups. All were administered orally once at a gavage volume of 10ml/kg body weight.

2. Sample source and processing:

1) Blank control group: physiological saline, NaCl content 0.9%.

2) Positive control group: Dissolve 0.2 g of Tankejing Powder in 10 ml of normal saline to obtain the positive control Tankejing solution with a concentration of 20 mg/ml.

3) Low-dose group of naringenin samples: Accurately weigh 5 mg of naringenin samples, use distilled water to make up the volume into a 10 ml volumetric flask, dissolve with ultrasonic waves for 5 minutes, and shake well to obtain the product. The concentration is 0.5mg/ml.

4) Medium-dose group of naringenin samples: accurately weigh 15 mg of naringenin samples, dilute to a 10 ml volumetric flask with distilled water, dissolve with ultrasonic waves for 5 minutes, and shake well to obtain the product. The concentration is 1.5 mg/ml.

5) High-dose group of naringenin samples: Precisely weigh 45 mg of naringenin samples, dilute to a 10 ml volumetric flask with distilled water, dissolve with ultrasonic waves for 5 minutes, shake well, and obtain. The concentration is 4.5 mg/ml.

6) Low-dose group of naringenin sodium salt sample: Accurately weigh 5 mg of naringenin sodium salt sample, dilute it to a 10 ml volumetric flask with distilled water, dissolve it with ultrasonic waves for 5 minutes, shake well, and obtain. The concentration is 0.5mg/ml.

7) Medium-dose group of naringenin sodium salt sample: accurately weigh 15 mg of naringenin sodium salt sample, dilute it to a 10 ml volumetric flask with distilled water, dissolve it with ultrasonic waves for 5 minutes, shake well, and obtain. The concentration is 1.5 mg/ml.

8) High-dose group of naringenin sodium salt sample: Accurately weigh 45 mg of naringenin sodium salt sample, dilute it to a 10 ml volumetric flask with distilled water, dissolve it with ultrasonic waves for 5 minutes, and shake well to obtain the product. The concentration is 4.5 mg/ml.

3. Experimental method:

1) Drawing of the standard phenol red curve: Accurately weigh a certain amount of phenol red with an analytical balance, dissolve it with 5% sodium bicarbonate, make it contain 12.5ng per 1ml, and then perform multiple dilutions sequentially to obtain phenol red content per ml 6.25ng, 3.125ng, 1.5625ng, 0.7813ng, 0.3906ng, 0.1953ng, 0.0977ng, 0.0488ng, OD value was measured with a spectrophotometer. Take the phenol red concentration as the ordinate and the OD value as the abscissa, and calculate the regression equation according to the phenol red concentration and OD value. According to the regression equation, the excretion of each mouse phenol red was calculated.

2) The mice were fasted without water for 12 hours.

3) Administration by intragastric administration. According to the order of animal numbers, each mouse was fed for 3 minutes after gavage, and then another mouse was fed with a time interval of 3 minutes. The total gavage time of 10 mice in each group was 30 minutes.

4) Half an hour after intragastric administration, each mouse was intraperitoneally injected with 0.2 ml of 5% phenol red saline solution. In order, each mouse was injected with phenol red 3 minutes after intraperitoneal injection, and then another mouse was injected, 10 mice for a total of 30 minutes.

5) Half an hour after the intraperitoneal injection of each mouse, the mice were sacrificed sequentially by dislocation of the cervical spine, and the time interval between sacrifices was 3 minutes. After the animal was sacrificed, it was fixed on the operating 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) Rinse the outer wall of the trachea with physiological saline drawn into a large syringe to wash away blood and phenol red in the outer wall of the trachea, and dry the washing solution with filter paper.

7) First cut off the trachea at the branch of the trachea, and then cut off the trachea at the upper end of the other end of the thyroid cartilage (including the cricoid thyroid cartilage).

8) Put each tracheal segment into a test tube filled with 1.5 ml of 5% NaHCO ₃ solution in advance.

9) Complete the above-mentioned trachea separation and shearing work within 3 minutes. Treat the second mouse in the same way. The method is as above.

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

11) Measure the OD value of the solution in each test tube at 546nm with a 721-type spectrophotometer.

12) Place the trachea section of each test tube 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 content.

14) Calculate the corrected phenol red content according to the phenol red content and animal body weight, and perform variance analysis with SPSS8.0 statistical software. Corrected phenol red content = phenol red content (ng)/mouse body weight (kg)

4. Experimental results:

According to the statistics, the output of phenol red in each dosage group is shown in the table below.

                 Table 2. Phlegm-reducing effects of naringenin and its salts (X±S)

Group

Phlegm reduction ^rateb

Dose Animal trachea phenol Corrected phenol red ^contenta

P-value

Code Name Sample Name (mg/kg) Red Content (ng) (ng/kg)

(%)

1 Normal saline 0 0.9385±0.4148 46.7282±22.6106 - -

2 Tankejing 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 1.9257±0.6713 102.7325±41.0313 P＜0.01 219.85

6 Naringenin sodium low dose group 5 1.3029±0.3069 69.4942±16.3724 P＜0.05 148.72

7 Naringenin sodium salt medium dose group 15 1.4225±0.7361 75.8773±39.2651 P＜0.05 162.38

8 Naringenin sodium high-dose group 45 1.9705±0.6898 105.1057±36.7945 P＜0.01 224.93

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

b: expectoration rate = drug treatment group / blank control group × 100%

Calculation method of phenol red content (ng): OD value × 7.6266-0.0554

From naringenin samples and the positive drug Tankejing, oral administration increases the bronchial secretion of mice, and it can be seen that the secretion of bronchial secretion of mice in low, middle and high dose groups of naringenin is different from that of the blank. Compared with the control group, there was a significant increase, and there was a statistically significant difference. Compared with the positive control drug Tankejing, the high-dose group has a significant increase in the bronchial secretion of mice, and there is a statistically significant difference, and the curative effect is more significant than the positive drug Tankejing. Naringenin sodium salt samples and the positive drug Tankejing, oral administration can increase the bronchial secretion of mice. Experiments show that the low, medium and high dose groups of naringenin sodium salt have different effects on the secretion of 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 Tankejing, the high-dose group has a significant increase in the bronchial secretion of mice, and there is a statistically significant difference, and the curative effect is more significant than the positive drug Tankejing. It shows that naringenin and/or naringenin salt have a good effect of relieving cough and reducing phlegm, and have obvious curative effects on diseases such as cough and phlegm caused by acute and chronic bronchitis and colds.

Embodiment 17: Toxicology experiment of naringenin drug

Under the temperature of 28±1° C. and the humidity of 70±5%, 20 healthy clean-grade NIH mice aged 7-8 weeks were selected, half male and half female, weighing 20-22 g. The feed and water were disinfected, and the animals were raised under normal feed conditions before the test and during the observation period of the test.

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 1, 4, 8, and 12 hours after administration, and observe every 12 hours thereafter. Mortality was observed, and body weight changes and other symptoms of the mice were recorded every day. On the 10th day, the mice were killed by neck dislocation, and the organs were 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. The pathological examination of each organ of the mice was normal, no pathological changes were found, and the weight of the mice did not lose weight within 10 days. Therefore, it shows that the naringenin drug of the present invention has no toxicity when it is orally administered to animals.

Embodiment 18: Naringenin capsule preparation

Prepare gelatin capsules according to the following ingredient proportions:

Naringenin 60

  Dried starch                              

  Micro-powder silica gel                            

Mix the auxiliary materials and naringenin evenly, put them into gelatin capsules, and get ready. Capacity: 100mg/capsule.

Example 19: Naringenin Tablets

Prepare tablets according to the following ingredient proportions:

Naringenin 500g

Starch 472.5g

  Starch Slurry (14%)                                                                                

Magnesium stearate 2.5g

Total 1000g

Mix naringenin and starch evenly, add starch slurry and continue stirring to make a soft material, granulate with a 10-mesh nylon sieve, ventilate and dry at 80-90°C, add magnesium stearate to the dry granules, pass through a 12-mesh sieve for granulation, and mix Evenly, pressed into tablets. A total of 10,000 tablets were obtained, each weighing about 0.1 g.

Embodiment 20: Naringenin inhalation

Prepare an inhaler according to the following composition ratio:

  naringenin                                                  

Lactose 500g

Poloxamer 1g

                                         

                                              

                                               

Total 911.5g

Dissolve naringenin, lactose, poloxamer and L-leucine in PEG400 aqueous solution, and then spray dry, add micronized silica gel to the obtained spray-dried powder, mix evenly, and pack in capsule-type dry powder inhalation device.

Claims (12)

1. naringenin and salt thereof are used for the application of the medicine of relieving cough and resolving phlegm in preparation.

2. according to the described application of claim 1, it is characterized in that said medicine contains the naringenin of 0.1-100%wt. or/and naringenin salt.

3. according to the described application of claim 1, it is characterized in that said medicine by simple naringenin or/and naringenin salt form, perhaps by as the naringenin of effective ingredient or/and naringenin salt and other effective ingredient or/and conventional pharmaceutical aids form.

4. according to the described application of claim 3, it is characterized in that said medicine is a capsule, its inclusions contains the naringenin of 0.5-100%wt. or/and naringenin salt.

5. according to the described application of claim 3, it is characterized in that said medicine is tablet or inhalant, its contain be no less than 0.1%wt. naringenin or/and naringenin salt.

6. according to the described application of one of claim 1～5, it is characterized in that said naringenin and naringenin salt obtain by chemical method is synthetic.

7. according to the described application of one of claim 1～5, it is characterized in that said naringenin is obtained through acid, alkali or enzyme hydrolysis by naringin crude product or monomer.

8. according to the described application of claim 7, it is characterized in that the concrete grammar of said naringin crude product or monomeric acid hydrolysis, basic hydrolysis or enzyme hydrolysis is: naringin crude product or monomer are dissolved in water, and stir, and add acid, alkali or enzyme, mix homogeneously; Mixed solution room temperature/heating hydrolysis; Hydrolysis finishes, and precipitation filters, and gets the naringenin crude product; Again through organic solvent or water recrystallization repeatedly, the naringenin monomer; Described be dissolved in water the water yield that adds by volume/weight ratio is naringin crude product or monomeric 1～1000 times; Acid in the acid hydrolysis can be hydrochloric acid, sulphuric acid, phosphoric acid or nitric acid, and the consumption of acid is to add the consumption that makes solution PH≤7 after the acid, and acid-hydrolyzed temperature is room temperature～100 ℃, and the time of hydrolysis is 0.1～170 hour; Alkali in the basic hydrolysis is sodium hydroxide, potassium hydroxide, calcium hydroxide, and perhaps organic base, the consumption of alkali are to add the consumption that makes solution PH 〉=7 behind the alkali, and the temperature of basic hydrolysis is room temperature～100 ℃, and the time of hydrolysis is 0.1～170 hour; Enzyme in the enzyme hydrolysis is the enzyme of various fracture hexoses, and the consumption of enzyme is that to add the concentration that makes enzyme in the solution behind the enzyme be 0.1 micromoles per liter～0.1 mole/milliliter, and the temperature of enzyme hydrolysis is room temperature～70 ℃, and the time of hydrolysis is 0.1-120 hour; The used organic solvent of recrystallization is ethanol, methanol, chloroform, ethyl acetate, acetone or petroleum ether, and the number of times of recrystallization is for once to ten times.

9. according to the described application of one of claim 1～5, it is characterized in that said naringenin is to extract naringenin crude product or the naringenin monomer that obtains from the medical material that contains naringenin.

10. according to the described application of claim 9, it is characterized in that the said concrete grammar that extracts naringenin from the medical material that contains naringenin is: pulverizing medicinal materials, one to multiple inferior through water/organic solvent extraction, filter merging filtrate; Filtrate is condensed into extractum, and extractum is placed precipitation, gets the naringenin crude product; Again through organic solvent or water recrystallization repeatedly, the naringenin monomer; The used organic solvent of described organic solvent extraction is ethanol, methanol, chloroform, ethyl acetate, acetone or petroleum ether, water or organic solvent extraction adopt under ℃ condition of room temperature～100 or/and carry out under the ultrasound wave condition, extraction time is one to three time, the consumption of water or organic solvent is not for having medical material at least, and each extraction time is 0.1-144 hour; The used organic solvent of recrystallization can adopt ethanol, methanol, chloroform, ethyl acetate, acetone or petroleum ether, and the number of times of recrystallization is for once to ten times.

11., it is characterized in that said naringenin salt is meant the slaine that naringenin and corresponding alkali are generated according to the described application of one of claim 1～5; The molecular formula of this naringenin salt is: C ₁₅H _nO ₅Y _m, n=9-11, m=1-3, Y are metal ion.

12. according to the described application of claim 11, it is characterized in that said naringenin salt is meant naringenin and sodium hydroxide or potassium hydroxide reaction and the naringenin sodium salt or the potassium salt that obtain, its molecular formula is: C ₁₅H _nO ₅Y _m, n=9-11, m=1-3, Y are K or Na ion.