CN101524397B - Licorice flavonoids antitussive and application thereof - Google Patents

Abstract

The invention provides an application of licorice flavonoids in preparing antitussives for treating acute and chronic cough caused by a plurality of reasons. The related acute and chronic cough comprises cough symptoms caused by various diseases such as acute and chronic tracheitis, asthma, chronic obstructive disease of lung, idiopathic pulmonary fibrosis, tuberculosis, bacterial pneumonia, bronchiectasis and the like. The licorice flavonoids extracted from liquorice proves to have very strong effect in preventing cough according to pharmacological tests; drug potency thereof is similar to the drug potency of codein in narcotic antitussive; the function mechanism analyses indicate that the licorice flavonoids plays the role of preventing cough through the non-narcotic nerve centre. In addition, the licorice flavonoids extracted from natural plants can also be prepared into health food. The invention solves the problem that the non-narcotic potent antitussives are badly needed, provides a non-narcotic potent antitussives which has fine social and economical benefits.

Description

Licorice flavonoids antitussive and application

Technical field

The invention belongs to pharmaceutical preparation and purposes, relate to the pharmaceutical preparation and the purposes of licoflavone, be specifically related to the application of licoflavone in the acute and chronic cough medicine that preparation treatment a variety of causes causes.

Background technology

In China, the Radix Glycyrrhizae medicinal history is long, is widely used.Traditional medicine thinks that it has effects such as QI invigorating invigorating middle warmer, heat-clearing and toxic substances removing, expelling phlegm for arresting cough, relieving spasm to stop pain, coordinating the actions of various ingredients in a prescription, has saying of " ten sides, nine grass ".Radix Glycyrrhizae is the root and the rhizome of pulse family (Leguminosae) Glycyrrhiza (GlycyrrhizaLinn.) various plants.There are 3 kinds according to its former plant of a record of Pharmacopoeia of the People's Republic of China version in 2005, i.e. Glycyrrhiza uralensis Fisch. G.uralensis Fisch., Glycyrrhiza inflata Bat. G.inflate Bat and Glycyrrhiza glabra L. G.glabraL..Along with the development of pharmacy and related discipline and research equipment, people are also more and more abundanter to the understanding of Radix Glycyrrhizae.Mainly contain chemical constituents such as glycyrrhizic acid, enoxolone, flavone, alkaloid and aminoacid in the Radix Glycyrrhizae, have physiologically active widely.Glycyrrhizic acid and enoxolone research in the past are more deep, but it is found that in recent years wherein flavones ingredient has stronger biological activity, have become new research focus.Up to now, from Radix Glycyrrhizae, isolate more than 150 flavone compound, can be divided into (CHINA JOURNAL OF CHINESE MATERIA MEDICA .2003 such as flavonoid, flavonols, osajin, chalcones, flavanone class, two hydrogen chalcones; 28 (7): 593-597).Licoflavone has been studied proof has antitumor action, antioxidation, anti-HIV effect, antiulcer action, antiarrhythmic effect and hepatoprotective effect etc.; But whether licoflavone can have the antitussive effect report very few, especially what is the material base of antitussive effect? What is the mechanism of action of antitussive effect? And preparing preparation with licoflavone does not see as yet that as the research of antitussive medicine report is arranged separately.

Factors such as along with development of global economy, industrial and civilian polluter constantly increases, and environmental improvement is ineffective in addition, the sickness rate of respiratory system disease is obvious ascendant trend, and its mortality rate that causes also rises year by year.But, this has created more vast market space also for the enterprise of production respiratory system disease medicine.Cough is one of cardinal symptom of respiratory system disease, and using cough medicine is the important means of symptomatic treatment.The rational Application cough medicine not only can alleviate patient's misery, and can prevent the generation of complication and the further deterioration of primary disease.In Chinese drug market, cough medicine was second largest OTC (OTC) medicine except that coldrex in 2004, and sales volume accounts for 5% of Pharmaceutical retail total value, and the annual market share is about 3,000,000,000 yuan.But the clinical practice cough medicine is mainly prescription drugs at present, accounts for 70%, is main with Western medicine, and strong cough medicine of imitating mainly is narcotic maincenter cough medicine (an anesthesia management medicine).Therefore, the strong effect cough medicine of searching and extraction non-narcotic has good social benefit and economic benefit from Chinese medicine.

Summary of the invention

The purpose of this invention is to provide the application of licoflavone in the acute and chronic cough medicine that preparation treatment a variety of causes causes.

Acute and chronic cough involved in the present invention comprises the cough symptom that various diseases such as acute and chronic tracheitis, asthma, chronic obstructive disease of lung, idiopathic pulmonary fibrosis, pulmonary tuberculosis, bacterial pneumonia, bronchiectasis cause, the improvement of the symptom that is used to cough.

Licoflavone of the present invention is an active constituents of medicine; Preparation process according to routine; Separately or add excipient substances such as conventional excipient, flavoring agent, antiseptic, lubricant, wetting agent, binding agent, thickening agent, solubilizing agent; Process any dosage form that is suitable for using clinically, like capsule, tablet, oral fluid agent etc.Specification is for to contain licoflavone 300mg by every (sheet).

Another object of the present invention provides the application of licoflavone in the health food of the acute and chronic cough symptom of preparation improvement.Licoflavone derives from natural plants, therefore, can licoflavone be processed health food, is used to improve the acute and chronic cough symptom that a variety of causes causes.

Usefulness of the present invention is: from the isolating licoflavone of Radix Glycyrrhizae, proved that through pharmacological testing licoflavone has very strong antitussive effect, its action intensity is close with narcoticness cough medicine codeine; These mechanism of action analyses show that licoflavone plays antitussive effect through the non-narcotic central action.Solve the problem of the strong effect cough medicine of present shortage non-narcotic, the strong effect cough medicine of non-narcotic is provided, had good social benefit and economic benefit.

Description of drawings

Fig. 1 is that licoflavone suppresses the dose-effect relationship that citric acid induces the guinea pig cough to reflect.

Fig. 2 is that licoflavone suppresses the dose-effect relationship that capsaicin induces the guinea pig cough to reflect.

Fig. 3 is that licoflavone suppresses the time-effect relationship that capsaicin induces the guinea pig cough to reflect.

Fig. 4 is that naloxone, ergometrine and glibenclamide suppress the influence that citric acid is induced guinea pig cough's reflection to licoflavone and codeine.

Fig. 5 is licoflavone is induced cough to electricity irritation cat superior laryngeal nerve a inhibitory action.

Fig. 6 is preparation technology's flow chart.

The specific embodiment

The present invention combines accompanying drawing and embodiment to be further described.

Embodiment 1

One, pharmacodynamics test

We at first simulate clinical cough symptom and have set up 2 kinds of guinea pig cough's animal models; Find the medicine that licoflavone of the present invention prepares after using this scale-model investigation; Dosage with 30mg/kg, 100mg/kg, 300mg/kg can suppress citric acid or the inductive guinea pig cough's reflection of capsaicin; Can block the inhibitory action that licoflavone induces the guinea pig cough to reflect to citric acid in advance lysergic acid amide (Methysergide, 5-hydroxytryptamine receptor blocking agent) 3mg/kg (ip); Slightly weaken the inhibitory action that licoflavone induces the guinea pig cough to reflect to citric acid in advance glibenclamide (Glibenclamide, the potassium channel blocking agent that ATP is responsive) 20mg/kg (ip).0.3mg/kg (ip) can not block the inhibitory action that licoflavone induces the guinea pig cough to reflect to citric acid to naloxone (Naloxone, opiate receptor blocking agent), and naloxone can be blocked the inhibitory action that licoflavone induces the guinea pig cough to reflect to citric acid.Be dosage dependence inhibition electricity irritation cat superior laryngeal nerve after licoflavone 10,30 and the 100mg/kg administration and cause coughre flex, average threshold voltage and matched group have improved 15%, 57% (P＜0.01) and 154% (P＜0.001) more respectively.These mechanism of action analyses show that licoflavone, licoflavone active component liquirtin, glycyrrhizin and liquirtin celery sugar play antitussive effect through the non-narcotic central action.Licoflavone, licoflavone active component liquirtin, glycyrrhizin and liquirtin celery sugar are wanted can be used for clinical treatment or improve the acute and chronic cough symptom that a variety of causes causes as non-narcotic maincenter antitussive.The specific embodiment is following:

One) licorice flavones preparation is to the effect and the mechanism of Cavia porcellus citric acid or the inductive guinea pig cough's reflection of capsaicin

1 experiment material and method

1.1 laboratory animal

The ZMU:DHP-1 Cavia porcellus, 300-400g, male and female are regardless of, and purchase the Experimental Animal Center in Zhejiang University, the quality certification number: 220010014.All operations and experiment flow are all in accordance with " management of laboratory animal regulations ".Animal is all ad lib and water inlets before experiment.

1.2 main agents and equipment licoflavone (being called for short LF) are extracted by this laboratory, measuring main component through HPLC is licoflavone, contains total flavones more than 50%, is main component with liquirtin and liquirtin celery sugar, does not contain glycyrrhizic acid; Citric acid, Shanghai chemical reagent factory, lot number: 20000121.Capsaicin (Capsaicin); Lot number: 123H78341, naloxone (Naloxone hydrochloride Dihydrate); Lot number: 446754/1, lysergic acid amide (Methysergide maleate salt), lot number: 073k1279 and glibenclamide (Glibenclamide), lot number: 123k1062; All purchase in Sigma Chemical Co. (St.LouisMissouri, USA).Codeine phosphate, Qinghai Pharmaceutic Plant, lot number: 220103.Instrument: plethysmograph, differential pressure transducer, computer (Pentium III, 128M/30G), MedLab bio signal acquisition processing system (MedLab V5.0.0, Nanjing Mei Yi company), PARI MASTER compression inhalation machine (PARI, MASTER; Germany).

1.3 method

1.3.1 citric acid induces the guinea pig cough to test 63 of (dose-effect relationship experiment) Cavia porcelluss, is divided into 6 groups at random, several 10～13 of every treated animal.Except that model control group and blank control group with the distilled water (ig); All the other each groups are licoflavone 30,100,300mg/kg (ig) respectively; Codeine (positive control drug) 10mg/kg (ig), 1h after the administration places 20 * 10 * 10cm plethysmography box with Cavia porcellus respectively; Import the pressure change signal in the container into microcomputer Medlab bio signal acquisition system with pressure transducer, trace respiratory wave and cough ripple.After Cavia porcellus is put into the plethysmography box and stablizes 1min, with nebulizer aerosol 15% liquor sodii citratis (blank control group is used physiologic saline for substitute) 30sec.The counting number of times of in aerosol finishes back 10min, coughing.Cough all to hear guinea pig cough's sound at every turn, see that seeing obvious respiratory wave on cough action and the microcomputer Medlab changes analysis-by-synthesis.

1.3.2 influence to guinea pig cough due to the capsaicin

1.3.2.1 81 of dose-effect relationship experimental guinea pigs are divided into 6 groups at random, several 12～18 of every treated animal.Except that model control group and blank control group with the distilled water (ig), all the other each groups are licoflavones 30,100,300mg/kg (ig) respectively, codeine (positive control drug) 10mg/kg, 1h after the administration places 20 * 10 * 10cm plethysmography box with Cavia porcellus respectively.After stablizing 1min, with the continuous aerosol 10min of nebulizer aerosol 0.015% capsaicin solution (blank control group is used physiologic saline for substitute).Counting begins to the cough number of times that finishes from aerosol.Cough all to hear guinea pig cough's sound at every turn, see that seeing obvious respiratory wave on cough action and the microcomputer Medlab changes analysis-by-synthesis.

1.3.2.2 69 of time-effect relationship experimental guinea pigs are divided into 7 groups at random, several 9～14 of every treated animal.Except that model control group with the distilled water (ig), each medication group licoflavone 100mg/kg (ig), 1h after the administration respectively, 4h, 6h, 8h, 12h places 20 * 10 * 10cm plethysmography box with Cavia porcellus.After stablizing 1min, with the continuous aerosol 10min of nebulizer aerosol 0.015% capsaicin solution (blank control group is used physiologic saline for substitute).Counting begins to the cough number of times that finishes from aerosol.Cough all to hear guinea pig cough's sound at every turn, see that seeing obvious respiratory wave on cough action and the microcomputer Medlab changes analysis-by-synthesis.

1.3.3 130 of licoflavone antitussive mechanism analysis Cavia porcelluss are divided into 8 groups at random, several 10～20 of every treated animal.Be divided into model control group, licoflavone group, naloxone+licoflavone group (N+LF group), ergometrine+licoflavone group (M+LF group), glibenclamide+licoflavone group (G+LF group), codeine (Cod) group, naloxone+Cod group (N+Cod group) and ergometrine+Cod organizes (M+Cod group).The licoflavone group, the N+LF group, the M+LF group, the G+LF group, the preceding 30min of LF100mg/kg (ig) (except the G+LF group, being 5min) is respectively with normal saline, naloxone 0.3mg/kg, ergometrine 3mg/kg or glibenclamide 20mg/kg (ip).Cod group, N+Cod group and M+Cod group, with normal saline, naloxone 0.3mg/kg and ergometrine 3mg/kg (ip), model group is used physiologic saline for substitute to the preceding 30min of Cod 10mg/kg (ig) respectively.1h aerosol citric acid draws and coughs behind the gastric infusion.Respectively Cavia porcellus is placed 20 * 10 * 10cm plethysmography box.After stablizing 1min, with nebulizer aerosol 15% citric acid soln 30sec.The counting number of times of in aerosol finishes back 10min, coughing.Cough all to hear guinea pig cough's sound at every turn, see that seeing obvious respiratory wave on cough action and the microcomputer Medlab changes analysis-by-synthesis.

1.4 result

1.4.1 guinea pig cough's due to the citric acid influence (dose-effect relationship) blank control group Cavia porcellus is given behind distilled water (ig) 1h with normal saline aerosol 30sec, and aerosol finishes not see that coughre flex is arranged in the back observation 10min; Model group aerosol 15% citric acid soln 30sec, counting average cough number of times in aerosol finishes back 10min reaches 28 times.Licoflavone 30,100 and 300mg/kg (ig) are dosage and rely on the coughre flex that the inhibition citric acid causes, licoflavone 100 and 300mg/kg and codeine 10mg/kg (ig) relatively antitussive effect intensity are suitable, referring to table 1 and Fig. 1.

Table 1. licoflavone suppresses the dose-effect relationship that citric acid induces the guinea pig cough to reflect.

Licoflavone and codeine are coughed preceding 60min (ig) in drawing. statistics: Student ' s t-test or Mann-Whitney Rank SumTest; Compare with model group ^*P＜0.01, ^{* *}P＜0.001; Compare with blank control group ^###P＜0.001.

1.3.4 influence (dose-effect relationship) the blank control group Cavia porcellus aerosol normal saline 10min to guinea pig cough due to the capsaicin does not see that Cavia porcellus has coughre flex; Model group aerosol capsaicin 10min, the number of times of on average coughing reaches 22 times.Licoflavone 30,100 and 300mg/kg ( Ig) be dosage and rely on suppress the coughre flex that capsaicin causes, licoflavone 300mg/kg and codeine 10mg/kg ( Ig) relatively antitussive effect intensity is suitable, referring to table 2 and Fig. 2.Licoflavone and codeine are coughed preceding 60min (ig) in drawing. statistics: mean ± SD, Student ' s t-test or Mann-Whitney Rank Sum Test; Compare with model group ^*P＜0.01, ^{* *}P＜0.001; Compare with blank control group ^###P＜0.001.

Table 2. licoflavone suppresses the dose-effect relationship that capsaicin induces the guinea pig cough to reflect

1.3.5 the persistent period of licoflavone antitussive effect (time-effect relationship) licoflavone 100mg/kg (ig) back 1h, three time points of 4h and 6h are compared with model group; The cough number of times obviously reduces; Wherein (ig) back 1h antitussive effect is the strongest, compares P＜0.01 with model group, referring to table 3 and Fig. 3.Statistics: mean ± SD, Student ' s t-test or the check of Mann-Whitney Rank Sum Test method. compare with model group ^*P＜0.05, ^*P＜0.01.

Table 3. licoflavone suppresses the time-effect relationship that capsaicin induces the guinea pig cough to reflect

1.3.6 ergometrine, naloxone and glibenclamide induce the ergometrine 3mg/kg that influences of guinea pig cough's reflection to compare with independent licoflavone in the preceding 30min of licoflavone 100mg/kg (ig) (ip) to licoflavone and codeine inhibition citric acid; Obviously reduce the suppression ratio of licoflavone; Significant difference highly significant (P＜0.001), the antitussive effect of prompting ergometrine licoflavone capable of blocking; Glibenclamide 20mg/kg slightly weakens the antitussive effect (see figure 4) of licoflavone in the preceding 5min of licoflavone 100mg/kg (ig) (ip); Naloxone 0.3mg/kg then can not block the antitussive effect of licoflavone in the preceding 30min of licoflavone 100mg/kg (ig) (ip).In contrast; Naloxone 0.3mg/kg obviously blocks the antitussive effect of codeine in the preceding 30min of codeine 10mg/kg (ig) (ip); Significant difference highly significant (P＜0.001); Ergometrine 3mg/kg also can reduce the suppression ratio (P＜0.05) of codeine in the preceding 30min of codeine 10mg/kg (ig) (ip), referring to table 4 and Fig. 4.Naloxone (0.3mg/kg) or ergometrine (3mg/kg) 30min lumbar injection before licoflavone and codeine administration, glibenclamide is the 5min lumbar injection before the licoflavone administration.Licoflavone and codeine are coughed preceding 60min (ig) in drawing.Statistical method: mean ± SD, Student-Newman-Keuls; ^*P＜0.05vs model group, ^{* *}P＜0.001vs model group; ^####P＜0.001vs LF group; ^▲P＜0.05, ^{▲ ▲ ▲}P＜0.001vs Cod group. table Chinese medicine name abbreviation: LF (licoflavone), N (naloxone), M (ergometrine), G (glibenclamide), Cod (codeine).

Table 4 naloxone, ergometrine and glibenclamide suppress the influence that citric acid is induced guinea pig cough's reflection to licoflavone and codeine.

1.5 discuss and brief summary

In the research in this stage, we have proved that licoflavone is dosage and rely on suppresses the coughre flex that citric acid and capsaicin are induced Cavia porcellus.Licoflavone 100mg/kg (ig) the antitussive effect persistent period is more than the 6h.Preliminary mechanism of action analysis and research proof, the antitussive effect of ergometrine licoflavone capable of blocking and the antitussive effect of partly blocking codeine, the antitussive effect of prompting licoflavone and codeine maybe be relevant with 5-hydroxy tryptamine (5-HT) approach.Kamei et al (1987) proves that the 5-HT level decline in the brain can weaken the action intensity of central antitussive, but periphery property antitussive is not had influence ^[1]Kamei et al (1988) is with 5, and (5,7-dihydroxytryptamine) the processing neonate rat makes 5-HT level decline 19% in the brain to 7-deshydroxy tryptamines, and the result causes codeine ultra quick to the inhibitory action of cough ^[2]In addition, 5, the rat that 7-deshydroxy tryptamines was handled, the antitussive effect that can be observed codeine obviously strengthens, but can partly be blocked by ergometrine.Therefore, the obvious enhancing of codeine antitussive effect possibly be because the change of 5-HT receptor sensitivity.These research promptings 5-HT receptor is playing an important role aspect the central antitussive activity, and with property cough medicine relation is little on every side.

Glibenclamide is the responsive K+ channel blocker of an ATP-, and the experiment proof is arranged, and it can block the antitussive effect of moguisteine (moguisteine, a peripheral non-narcotic cough medicine) ^[3,4], can not block codeine and right romilar (dextromethorphan), the prompting moguisteine is not the cough medicine of a central, its action pathway is relevant with the responsive K+ passage of ATP-.In view of the above, people such as Kamei thinks that the responsive K+ passage of peripheral non-narcotic cough medicine mechanism of action and ATP-is relevant.In this experiment, the antitussive effect of our glibenclamide blocking-up licoflavone on probation, after the result showed the responsive K+ carrier frequency channel break of ATP-, the antitussive effect of licoflavone had slightly and weakens, but statistics no significant difference (see figure 4).The antitussive effect that glibenclamide fails to block licoflavone is not to mean that the antitussive effect of licoflavone and the responsive K+ passage of ATP-are irrelevant.With compare through the 5-HT receptor, the antitussive effect of licoflavone is stronger through the responsive K+ passage of ATP-generation antitussive effect, this experiment glibenclamide fails to block the antitussive effect of licoflavone maybe be relevant with used dosage.Because people's such as Kamei experiment shows glibenclamide and can block the inhibitory action (result is unexposed) that licoflavone induces the guinea pig cough to reflect to capsaicin, and this test employing is the inductive guinea pig cough's reflection model of citric acid.

In order further to understand the mechanism of the antitussive effect of licoflavone; We try out the antitussive effect of naloxone (an opiate receptor blocker) blocking-up licoflavone, and the result shows, does not influence the antitussive effect intensity of licoflavone after the opiate receptor blocking-up; But the antitussive effect of codeine capable of blocking; These results suggest, licoflavone are not through opiate receptor generation antitussive effect, are the cough medicines of a non-narcotic.

To sum up the result shows, licoflavone possibly be a central antitussive that effect is stronger, and its mechanism of action relates to the 5-HT approach, but property antitussive effect is machine-processed around can not getting rid of it.

Two) licoflavone is induced the influence of cough to electricity irritation cat superior laryngeal nerve

1 materials and methods

1.1 material

1.1.1 animal: cat, body weight 3.0 ± 1.0kg, male and female are regardless of.Purchase Experimental Animal Center, the quality certification number: 20040516 in Medical College of Zhejiang Univ..

1.1.2 main agents

(1) licoflavone (being called for short LF) is extracted by this laboratory, and measuring main component through HPLC is licoflavone, contains total flavones more than 50%, is main component with liquirtin and liquirtin celery sugar, does not contain glycyrrhizic acid.Become concentration with the normal saline preparation and be respectively 100mg/ml, 30mg/ml, the solution for standby of 10mg/ml;

(2) codeine phosphate tablets: codeine phosphate, Qinghai Pharmaceutic Plant, lot number: 220103.It is subsequent use as the 5mg/ml suspension to use physiological saline solution to be mixed with concentration;

(3) pentobarbital sodium: lot number: F20030816, China Medicine's (import packing), using normal saline to be mixed with concentration is 3.5% solution for standby.

1.1.3 key instrument

(1) MedLab-U/4C bio signal acquisition processing system, Meiyi Science & Technology Co., Ltd., Nanjing;

(2) electronic balance: Shanghai balance equipment factory, FA1104 type;

(3) electrostimulator: Model:SEN-3301, separator: Model:SS-202J, Nihon KohdenCorporation, Japan.

2. method

(1) blank control group 2.1 divide into groups: normal saline 1ml/kg gastric infusion, n=10; (2) positive controls: codeine phosphate 5mg/kg gastric infusion, n=10; (3) high dose group: licoflavone 100mg/kg gastric infusion, n=10; (4) dose groups in: licoflavone 30mg/kg gastric infusion, n=10; (5) low dose group: licoflavone 10mg/kg gastric infusion, n=10.

2.2 method

With pentobarbital sodium 35mg/kg intraperitoneal injection of anesthesia, back of the body position is fixed on the operating-table, cuts off the cervical region hair with cat; Along neck midline incision skin; Separate subcutaneous tissue, expose thyroid cartilage, find out a side vagus nerve; Find out the knot neuroganglion along the vagus nerve head-end, neuroganglion is told in the visible superior laryngeal nerve.The careful superior laryngeal nerve that separates is set up shield electrode, drips several drop of liquid paraffin at the discharge electrode place, in case dry.Separate trachea, with Y-piece circulation of qi promoting cannula, an end connects pressure transducer and is connected in MedLab-U/4C bio signal acquisition processing system record status of cough.Regulate electrostimulator, parameter is: Duration 0.5ms, maininterval 20ms; Interval 10ms, delay 1ms stimulates output voltage to increase progressively from low to high at every turn; Stimulate superior laryngeal nerve to cause the voltage threshold of cough before measuring administration; That surveys stimulates for 3 times the magnitude of voltage that can cause the cat cough to be drawing of this cat to cough voltage threshold continuously, then by the grouping situation to the cat gastric infusion, beginning behind the administration 30min stimulates.Every separated 5min stimulates once again, measures the situation of change of administration threshold value after 1 hour.

3, result

Visible from table 1, each treated animal electricity irritation cat superior laryngeal nerve before administration causes that the average threshold voltage of cough is between 0.64～0.70, no significant difference (P＞0.05) through between each group of statistics.Be dosage dependence inhibition electricity irritation cat superior laryngeal nerve after licoflavone 10,30 and the 100mg/kg administration and cause coughre flex, average threshold voltage and matched group have improved 15%, 57% (P＜0.01) and 154% (P＜0.001) more respectively.Show very strong antitussive effect after the positive control drug codeine 5mg/kg administration; Average threshold voltage and matched group have relatively improved 296% (P＜0.001); The difference (P＜0.001) that highly significant is more all arranged with three dose groups of licoflavone respectively; Effect obviously is better than licoflavone, sees Fig. 5 and table 5.Statistical method: mean ± SD, t-test; ^*P＜0.01vs matched group, ^{* *}P＜0.001vs matched group; LF: licoflavone.

Table 5 licoflavone to electricity irritation cat superior laryngeal nerve induce cough influence (x ± SD, n=10)

Annotate: compare with control group, ^{* *}P＜0.01, ^{* *}P＜0.001.

4. discuss and brief summary

Our verified licoflavone is dosage and relies on and suppress the coughre flex that citric acid and capsaicin are induced Cavia porcellus before.Licoflavone 100mg/kg (ig) the antitussive effect persistent period is more than the 6h.Preliminary mechanism of action analysis and research proof, the antitussive effect of 5-hydroxy tryptamine (5-HT) receptor antagonist ergometrine licoflavone capable of blocking, the antitussive effect of prompting licoflavone maybe be relevant with 5-hydroxy tryptamine (5-HT) approach.Kamei et al (1987) proves that the 5-HT level decline in the brain can weaken the action intensity of central antitussive, but periphery property antitussive is not had influence ^[4]Kamei et al (1988) is with 5, and (5,7-dihydroxytryptamine) the processing neonate rat makes 5-HT level decline 19% in the brain to 7-deshydroxy tryptamines, and the result causes codeine ultra quick to the inhibitory action of cough ^[5]5, the rat that 7-deshydroxy tryptamines was handled, the antitussive effect that can be observed codeine obviously strengthens, but can partly be blocked by ergometrine.In coughre flex that the cat superior laryngeal nerve that electricity irritation causes causes experiment, thing 5-hydroxytryptophan 5mg/kg (iv) not only suppresses respiratory frequency but also suppresses the coughre flex that cat superior laryngeal nerve that electricity irritation causes causes before the 5-HT ^[2], on the contrary, coughre flex and respiratory frequency that the cat superior laryngeal nerve that ergometrine 3mg vertebral artery interior injection can increasing electricity irritation causes causes ^[3]The rising of 5-HT level is shaped on inhibitory action to the maincenter generator of coughre flex in these research prompting brains, and the 5-HT receptor is playing an important role aspect the central antitussive activity.In the test in this stage, we have proved that further licoflavone can suppress the coughre flex that cat superior laryngeal nerve that electricity irritation causes causes, the main effect of prompting licoflavone possibly be to improve 5-HT level in the brain, thereby brings into play its maincenter antitussive effect.

Embodiment 2 licorice flavones preparations

1 prescription (licoflavone capsule)

Licoflavone 300g

Starch 32g

Hydroxypropyl cellulose (L-HPC) 6g

Micropowder silica gel 4.5g

Magnesium stearate 1.5g

Starch slurry (10%) is an amount of

Process 1000

The screening process of 2 prescription foundations and prescription

2.1 prescription foundation

Work out licoflavone capsule, specification: licoflavone content 300mg/ grain according to pharmacodynamics test.

Be corn starch among the starch we, color and luster is good, and a little less than the hygroscopicity, output is big, and price is low; Be white fine-powder, water insoluble and ethanol, very stable in air, inoperative with most drug, moisture absorption and deliquescence not, water-swellable is diluent and disintegrating agent the most widely, and is main as the diluent disintegrating agent among the we.

These article of hydroxypropyl cellulose (L-HPC) are white or white or crystalline powder, but in water insoluble but water absorption and swelling because the L-HPC powder has very big specific surface area and porosity,, increased dilatancy so bigger rate of moisture absorption and water absorption are arranged.These article consumption generally can be about 1%-5%, and consumption is 2% among the we.

Micropowder silica gel, these article are light-weight white powder, and odorless, tasteless is water insoluble and sour; Chemical property is stable, does not react with most medicines, and good flowability has bigger absorption affinity to medicine; Hydrophilicity is strong, helps the absorption of medicine, and these article consumption generally is merely 0.15%-3%; Because the principal agent licoflavone is mobile poor, be used to improve particulate flowability among the we, consumption is 1.5%.

Magnesium stearate is a white powder, and fine and smooth easily have good tack, is evenly distributed after granule mixes and not easily separated, only can demonstrate good lubrication on a small quantity, and general consumption is 0.3%-1%, and consumption is 0.5% among the we.

2.2 the screening process of prescription:

Design following three prescriptions (1000) specification: 300mg/ grain table 6-1

According to above three prescription pilot samples, and carry out above prescription screening test.

(1), carries out the dissolution test

1., assay method

Press second licoflavone capsule of Chinese Pharmacopoeia version in 2000 dissolution method (appendix XC second method) operation, (get anhydrous sodium acetate 82g and add water 7500ml, with glacial acetic acid pH value to 5.0 with the 0.1mol/L acetate buffer; Add water and make into 10000ml) be solvent; Rotating speed is that per minute 50 changes, and operation is in accordance with the law got solution on time and filtered through 0.8 μ m microporous filter membrane; Get subsequent filtrate and measure trap at the 482nm place, calculate the stripping percentage rate.

2., dissolution determination result

Get test sample respectively, press assay method and measure dissolution, sample time 5,10,15,20,25,30,45,60min, the result sees table 6-2.

Three prescriptions of 6-2 dissolution determination result

Corresponding dissolution result sees table 6-3 according to last table.

Table 6-3 dissolution determination result (n=6 X ± S)

The result shows: prescription 3 can reach 86.8%, 30 minute dissolution at 20 minutes dissolutions and almost reach maximum.Dissolution was 85% when prescription 1, prescription 2 only had 60 minutes 60%, 30 minute at 20 minutes absorbances.Reach 80% according to Chinese Pharmacopoeia dissolution 30 minutes the time, they all meet the pharmacopeia requirement.

This shows that the consumption of low-substituted hydroxypropyl cellulose (L-HPC) is merely 2% in the prescription 3, dissolution rate adds after the L-HPC among the visible we 3 obviously faster than prescription 1, prescription 2, helps capsular disintegrate and has accelerated the stripping of principal agent licoflavone.

(2), test angle of repose:

1,2,3 process granule by prescription, flow down through the long neck funnel of bore 7cm, and be cone shape, survey its angle of repose, the result sees table 6-4.

Table 6-4

2,3 angle of repose, the good fluidity that prescription 2,3 is described was in prescription 1 less than prescription 1 it is thus clear that write out a prescription.

Conclusion: micropowder silica gel can significantly improve particulate mobile performance.

In sum, the dissolution of prescription 3, good fluidity are in prescription 1,2, so selected prescription 3.

3 production technologies

3.1 method for preparing

Method for making: the licoflavone, starch and the L-HPC that take by weighing recipe quantity mix, and cross 60 purposes sieve three times, mix homogeneously; The starch slurry of adding 10% is made soft material in right amount, granulates with 16 orders, and 60 ℃ of dryings behind 16 order granulate, add micropowder silica gel, magnesium stearate mix homogeneously, process 1000 with No. 0 capsule.

3.2 the screening of process conditions

Manufacture experimently 1000 samples by prescription.

3.2.1 bulk density test

The granule of trial-production is packed in the 100ml graduated cylinder, fall twice with certain height, (each conservation condition is consistent, and degree of tightness is suitable, claims that its weight calculates its bulk density, and the result sees table 7-1.

Table 7-1 bulk density test data (n=3)

Therefore, can know according to above data that the average bulk density of granule is 0.46g/ml, every loading amount requires for 30mg, so select capsule No. 0.

3.2.2 wettability test

Investigate ambient humidity to influence degree in the capsule filling process, measured particulate hygroscopicity for this reason.

Take by weighing 12 parts of granules, every part of about 2g, the accurate title, decide, and was placed on different relative humidity environment held 7 days, surveys its weight change, and the result sees table 7-2.

Table 7-2 hygroscopicity determination data table (n=2)

It is thus clear that relative humidity particle weight under the 40-90% condition does not have to change basically, hygroscopicity does not obviously increase.Therefore, the relative humidity that can confirm this kind requires not strict, under general production environment, can produce.Can not impact pharmaceutical properties and stability because of moisture.

4 technological processes are referring to Fig. 6.

Two, licoflavone Soft Release tablet recipe and preparation technology

1 preparation prescription is formed

1.1 per 1000 contain following material (300mg/ sheet)

Liquirtin celery sugar 300g

Stearic acid 40g

HMPC 200g

Polyvinylpyrrolidone (K30) 80g

30% acrylic resin IV number an amount of

1.2 coating fluid prescription

(Y-1-7000) 60g (off-white color)

Ethanol 1000ml

2. preparation technology

2.1 the preparation of binding agent

Precision take by weighing acrylic resin IV number an amount of, using 95% ethanol to be mixed with concentration is 30% solution, subsequent use.

2.2 the preparation of label

Cross 60 mesh sieves after getting recipe quantity principal agent licoflavone and adjuvant stearic acid, HMPC, the abundant mixing of polyvinylpyrrolidone; Add 30% acrylic resin IV number in right amount, process and do wet moderate soft material, 24 mesh sieves are granulated; Dried 30 minutes for 50 ℃; 24 mesh sieve granulate, after the mensuration granule content was qualified, tabletting promptly got.

2.3 coating

2.3.1 the foundation of coating

The licoflavone slow releasing tablet is a Film coated tablets, considers factors such as attractive in appearance simultaneously, and these article are processed Film coated tablets.

2.3.2 the preparation of coating solution

Take by weighing (Y-1-7000) an amount of, slowly add in 70% the alcoholic solution, be mixed with 6% coating solution, on magnetic stirring apparatus, ceaselessly stir, until dissolving fully, promptly get.

Put in the coating pan 2.3.3 get plain sheet, pot Revolution Per Minute 30～40 changes, and kettle temperature is 40～50 ℃, uninterruptedly sprays on coating solution to the plain sheet, until evenly wrapping the thin film clothing, is drying to obtain.

2.4 the mensuration of critical relative humidity

At a certain temperature, it is an amount of to get the granule that makes by prescription and technology, is placed on respectively in the different saturated salt solutions; Place after 5 days, taking-up is weighed, and makes abscissa with the relative humidity of different saturated salt solutions; The granule percentage rate that in different saturated salt solutions, increases weight is made vertical coordinate, makes curve chart, and makes the tangent line of curve; The intersection point of gained two tangent lines is a critical relative humidity, and to draw critical relative humidity from figure be 74%.Measure the result 8-1 that sees the following form.

The mensuration of table 8-1 critical relative humidity

Relative humidity	9％	32.5％	66％	75％	92.5％
						Saturated salt solution	H 3PO 4	CaCl 2	NaNO 2	NaCl	KNO 3
Particle weight (g)	0.0345	0.0939	0.2187	0.2868	0.9182
						Weightening finish percentage rate (%)	0.2350	0.9067	2.168	2.848	9.102

The particulate critical relative humidity of these article is 74% (＞50%), explains that these article granule is non-hygroscopic under air-proof condition.

3. prescription foundation

3.1 confirming of dosage

Its specification of licoflavone slow releasing tablet is the 300mg/ sheet, and these article conventional amount used clinically are 300～900mg every day, when confirming the dosage of these article, and considers and makes things convenient for patient to take, and is convenient to dosage adjustments, contains licoflavone 300mg so confirm as every.

3.2 prescription screening

3.2.1 the interference of adjuvant test

Being chosen in the 333nm wavelength does not have and absorbs interferential adjuvant such as HMPC, polyvinylpyrrolidone, dextrin, starch, mannitol, stearic acid, acrylic resin etc.; Get a slice amount adjuvant by the prescription proportioning; Add in the container according to method under the assay item; Measure the trap of volumetric soiutions, experimental result is illustrated in 333nm wavelength adjuvant does not have absorption.

3.2.2 prescription screening process

The licoflavone water soluble discharges in order to make drug slow, and effective blood drug concentration can be kept the long period, and to reach therapeutic purposes preferably, therefore, we process the licoflavone slow releasing tablet, are matrix type.Domestic slow-release material commonly used has at present: polyvinylpyrrolidone, ethyl cellulose, stearic acid, hypromellose, acrylic resin etc.Contain licoflavone 30mg with every; Adopt a kind of in the above-mentioned controlled-release material or several and different size, different amounts prescription; Carry out the test of release in vitro degree, find that from more than ten slow release prescriptions 1, No. 2 prescription slow release effect is better, but No. 2 prescription release in vitro degree are best; And the repeatability of particulate compressibility, flowability and prescription is all good, so confirm that prescription 2 is this sustained-release tablet recipe.

The prescription screening process 3-2 that sees the following form:

Table 3-2

The prescription number	Prescription is formed (1000 amounts)	Criterion	The result
				1	Licoflavone 300g stearic acid 100g HMPC 150g acrylic resin II 20g 20% (K90) is an amount of	Hardness: 9kg angle of repose: 26 ° of outward appearances: still can degree of release 2 hours: 41.9% 6 hours 74.5% 12 hours: 98.5%	The release degree is slightly fast, and this prescription is infeasible.
2	Licoflavone 300g stearic acid 40g HMPC 200g (K30) 80g acrylic resin 5 is an amount of	Hardness:: 10kg angle of repose: 25 ° of outward appearances: degree of release is 2 hours well: 39.7% 6 hours 66.2% 12 hours: 87.1%	The release degree is good, compressibility, flowability, outward appearance etc., and this prescription is feasible.
				3	Licoflavone 300g acrylic resin II 20g dextrin 20g (K90) 20g stearic acid 50g ethyl cellulose is an amount of	Hardness:: 10kg angle of repose: 27 ° of outward appearances: degree of release is 2 hours well: 74.3% 6 hours 93.5% 12 hours: survey	The release degree is too fast.This prescription is infeasible.

3.2.3 three lot sample article of trial-production detect the result

Prescription and preparation technology according to confirming have manufactured experimently three lot sample article, and testing result is following:

Table 8-3

Lot number	Content %	Release degree %
			080219	98.79	2 hours: 40.5 6 hours: 63.9 12 hours: 85.3
080221	99.51	2 hours: 38.4 6 hours: 64.7 12 hours: 83.3

080223

99.63

2 hours: 38.9 6 hours: 62.6 12 hours: 82.6

Show from three batches of pilot sample testing results: prescription and preparation technology that these article are confirmed are feasible.3.2.4 with the phosphate buffer of pH6.8 release medium as these article; Method adopts the basket method (two appendix XC first methods of Chinese Pharmacopoeia version in 2000) of changeing; Rotating speed is that per minute 100 changes; Sample point is 1 hour, 3 hours and 10 hours, and we have redeterminated the release degree of three lot sample article and import sheet in view of the above, and the result is following:

Table 8-4

Lot number	Release degree %
		080219	1 hour: 31.43 hours: 54.410 hours: 85.8
080221	1 hour: 32.13 hours: 54.410 hours: 85.9
		080223	1 hour: 32.13 hours: 54.110 hours: 84.1

Sample (080219) to trial-production has carried out influence factor's test (exposure experiments to light, hot test, high wet test) simultaneously, and method and result are following:

(1) exposure experiments to light

At ambient temperature, these article are removed outer package sample are divided in plate, in the detection of taking a sample after 5,10 days of 4500lux light intensity held.The result sees table 8-5.

(2) hot test

These article are removed the listing packing, and sampling detects after 5,10 days respectively at placing in 60 ℃ the baking oven.The result sees table 8-6.

(3) high wet test

These article removal listing packing is put into the constant humidity vessel respectively, in 25 ℃, relative humidity 92.5% uncovered placement examination sampling detection after 5,10 days.The result sees table 8-7.

Table 8-5 exposure experiments to light result

^*Refer to the label color and luster, the equal no change of film-coat (down together)

Table 8-6. hot test result

Table 8-7. high humility result of the test

4. the effect of each adjuvant in prescription

4.1A: have hydrophobicity, stop the release of medicine.

4.2 hypromellose: be the hydrophilic macromolecule cellulosic material, meet water and form gel, reduce the speed that water permeates in sheet, delay the release of medicine, have slow releasing function.Be widely used in sustained-release preparation both at home and abroad.

4.3 polyvinylpyrrolidone: be hydrophilic high molecular material, meet water and form gel, reduce the speed that water permeates in sheet, delay the release of medicine, have slow releasing function.Be widely used in sustained-release preparation both at home and abroad

4.4 acrylic resin: commodity are called " Eudragit "; Because of its substituent group difference is divided into various models; Be widely used for the material as controlled slow-release preparation abroad, also as enteric coating material, this prescription selects it as binding agent; Utilize its film property packaging medicine, stop medicine in the gastric rate of release.

4.5B: be lubricant.

4.6Opadry: film-coat material, Main Ingredients and Appearance are hypromellose.

5. supplementary material is originated and quality standard

5.1 licoflavone: this laboratory extracts purification, and content purity is more than 99%.

5.2A: meet Chinese Pharmacopoeia version in 2005, Shanghai Yan'an Oil Chemical Plant produces.

5.3 hypromellose: meet American Pharmacopeia (USPXXIII), provide by Shanghai Colorcon company.

5.4 polyvinylpyrrolidone: meet American Pharmacopeia, the Shenyang Dongbei Pharmaceutical General Factory is produced.

5.5 acrylic resin IV: meet Chinese Pharmacopoeia version in 2005, produce by Lianyun Harbour system iodine factory.

5.6B: meet Chinese Pharmacopoeia version in 2005, produce by Fructus Vitis viniferae sugar refinery, Shanghai.

5.7Opadry: meet British Pharmacopoeia, provide by Shanghai Colorcon company.

Claims (4)

1. the application of licoflavone in preparation non-narcotic central antitussive thing.

2. the application of licoflavone in the health food of preparation non-narcotic central antitussive effect.

3. application according to claim 1; It is characterized in that: said medicine be active component with the licoflavone; Separately or one or more excipient substances that add in conventional excipient, flavoring agent, antiseptic, lubricant, wetting agent, binding agent, thickening agent or the solubilizing agent process, the preparation specification is for containing licoflavone 300mg by every or sheet.

4. application according to claim 3 is characterized in that: said preparation formulation is capsule, tablet.

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