The Study Of HaoHongfang On Anti-inflammatory Cough And The Mechanism

1,Objective:Due to population density, increasing smoking crowd, environmental pollution and other factors, research has shown that the incidence of respiratory disease at any age group account for the first of many diseases. The medical treatment of upper respiratory tract infection are the expectorant, antitussive and anti-infection and other basic methods. Chemicals, mainly cough, phlegm and other and other symptomatic treatment based, main effect rapidly, but antitussive drugs have side effects and easy to relapse after drug withdrawal, leading to extended refractory cough bound; with the increasing antibiotic resistance, the application of anti-infectives is restricted. Traditional Chinese medicines have greater advantages, clinical curative effect, which is not only bactericidal, antibacterial, antiviral and anti-inflammatory, but also expectorant, cough, asthma, and improve immunity, eliminate or alleviate the symptoms of pathogenic factors through an integrated role, but many of them have slow onset, taking the characteristics of inconvenience. So, the Development of a safe, quick, targeted, curative effect of Chinese medicine preparations which learn the advantages of Chinese and Western medicine is extremely urgent.It is the needs of clinical and patients, is also purpose of this topic to develop an unique therapeutic prescription for the southern district of fengreganmao and viral influenza. This topic is from many years of clinical experience. Haohongfang which can suppressing cough,anti-inflammatory,relieving asthma,is a traditional Chinese medicine compound of Modern extraction and purification of Artemisia annua and Folium. Firstly, the topic clarified the rationality of prescription through increased capillary permeability in mice the impact test, CBS spray induced cough cough impact tests on guinea pigs to clarify the prescription to the rationality, secondly selected the rational compatibility of the composition of Haohongfang, thirdly, study the cough and anti-inflammatory pharmacodynamics of Haohongfang and investigate the mechanism of it preliminary.2,Methods: 2.1 Study of Compatibility Rationality2.1.1 The impact on the capillary permeability in mice70 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 7 groups,10 mice/group named control group, Artemisia group, Folium group, compound group 1, compound group 2, compound group 3, aspirin group. Each treatment group were orally administered by 20 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/10g weight, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.2.1.2 Drugs on cough in guinea pigsHartley guinea pigs,250g or about, female and male in half, adapted to the environment for three days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution, spray equipment, to occur within 150 s of asthma (in convulsions, fall time as the incubation period) or cough times within five minutes extra in the 10 who qualified for sensitive animals.80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Folium group, Artemisia annua group, compound group 1, compound group 2 and compound group 3. Each treatment group were orally administered by 10 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.2.2 HaoHongfang on antibacterial, antiviral research2.2.1 HaoHongfang on the treatment in mice infected with Streptococcus pneumoniae200 SPF level NIH mice, male and female in half,18～22 g, adapted to the environment for three days.And then divided randomly into 7 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang high, medium and low dose group. Each treatment group was orally administered by 20 ml/kg, once a day,7 days in all; control group and model group were given equal volume of sesame oil.1 h after the last administration, intraperitoneal injection of Streptococcus pneumoniae (strain No. 31001-20) suspension, each 1.0 ml (0.6 billion bacteria/ml), blank control group and the control group, intraperitoneal administration of the injection volume of distilled water.1,6 h after infection and then administered once a week to observe and record the number of deaths in each group, compute the mortality rate. And compared the difference between the groups.2.2.2 HaoHongfan on the mortality rate of mice infected with Staphylococcus aureus200 SPF level NIH mice, male and female in half,18 to 22 g, after three days of adaptation to the environment were randomly divided into 8 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day for 7 days, control group and model group filling the same dose of sesame oil.1 h after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice were injected suspension of Staphylococcus aureus (strain No.26112-5), each 1.0 ml (lbillion bacteria/ml).1,6 h after the first infection were repeated dose 1, observe and record animals in each group died within a week the number of animal mortality calculated. And compared the difference between the groups.2.2.3 HaoHongfang on the infection of influenza virus in mice(1) virus inoculation Methods:9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 d at 4℃, the allantoic fluid samples for hemagglutination test, measuring hemagglutination titer, take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.(3) infection Mouse:SPF level obtained NIH mice, weighing 13～15g, male and female in half, to adapt to the environment in the laboratory after 3 days were randomly divided into 8 groups, the control group, model group, ribavirin Group Qingrexiaoyan Ning Group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, the mice in the light ether anesthesia, will have a good virus dilute solution (4 LD50/0.05 ml) intranasally infected mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day for 7 days, and daily records of animal morbidity and mortality, continuous observation for 14 days. Calculate the mortality rate and prolong life protection rate and compared the difference between the groups.2.2.4 HaoHongfang on Lung-index of mice infected with influenza virus(1) virus vaccination:from 9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 days at 4℃, the allantoic fluid samples for hemagglutination, hemagglutination titer test, to take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.(3) infection Mouse:SPF level obtained NIH mice,130, weight 13-15g, male and female in half, to adapt to the environment in the laboratory after 3 days, were randomly divided into 8 groups, the control group, model group, ribavirin group, Qingrexiaoyanning group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice in the light ether anesthesia, will have a good virus dilute solution (15 LD50/0.05 ml) intranasally infected with small mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day,4 consecutive days. Fasting before the last administration, mice can not help but water 8h, weighing, 1h after the last administration, animals were sacrificed and dislocation of cervical vertebra, quickly remove the entire lung, in normal saline wash 2, removal of the trachea, hilar lymph nodes and other organizations, the filter paper suction Dry the surface moisture, and weighed. Lung index and the inhibition rate calculated to compare the differences between each group.2.2.5 HaoHongfang on the death in mice induced by endotoxin120 SPF level NIH mice,18～22 g, male and female in half, adapted to the environment in the laboratory for 3 days, randomly divided into six groups, control group, model group, Qingrexiaoyanning Group HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day,7 days in all, control group and model group were given equal volume of sesame oil.1 h after the last administration, in addition to the blank control group were outside the remaining mice were injected D-GALN 200 mg/kg and LPS 1.5 mg/kg, control group, intraperitoneal dose of saline, observed 72 hours, record the number of animal deaths. There are differences between each group.2.3 HaoHongfang on clearing heat and relieving cough and phlegm2.3.1 HaoHongfang on fever induced by dry yeast60 qualified animals were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group.6h fasting before the experiment can not help but water. Control back of the neck rats injected with 10 ml/kg; 20% of the yeast suspension were injected 10 ml/kg in the back of rats and normal saline with control group. Modeling the same time each dose group were administered orally by dose and blank control group was given the sesame oil.1,2,3,4 hours after the drug 1 of each measured body temperature, body temperature decreased in all groups compared the value of the difference.2.3.2 HaoHongfang on endotoxin fever of rabbits36 qualified New Zealand rabbits were randomly divided into 6 groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. In addition to control group and model group, other groups in accordance with the dose administered,10 ml/kg, once a day,7 days in all, control group and model group were given the same dose of sesame oil. 1h after the last administration,10μg/ml·kg of endotoxin were injection from the rabbit ear vein,1,2,3,4,6 h after the administration temperature were measured one time,and compared the difference between each gtoup.2.3.3 HaoHongfang on cough in mice with ammonia waterSPF level NIH mice,18～22 g, female and male in half, randomly divided into 7 groups,15 in each group. Control group, respectively, codeine, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, given control group sesame oil.20 ml/kg, once a day,7 days in all.1 h (codeine 30 min) after the last administration, put constant pressure of guinea pigs by ammonia spray device (pressure of 140mmHg, ammonia concentration of 28%), ammonia by the constant pressure spray equipment fog (to be replaced before each mouse into the same volume of fresh ammonia solution), ammonia spray time, increasing by geometric progression, according to sequential changes in ammonia spray time to observe and record the mice receiving spray from fog began to cough occurred the time to calculate the time mice were half of the cough (EDT50), calculated according to R value (antitussive effects:R value> 130%, significant antitussive effect:R values> 150%). There are differences between each group.2.3.4 HaoHongfang on the citric acid spray lead cough cough of guinea pigsHartley guinea pigs,250～280g, female and male in half, adapted to the environment in the laboratory for 3 days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution spray equipment to 150s appeared in asthma (in convulsions, fall time as the incubation period) or 5 the number of extra minutes to cough 10 times the sensitivity of animals to qualified persons.80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, control group was given sesame oil,10 ml/kg, once a day,7 days in all. the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.2.3.5 HaoHongfang on the mouse trachea phenol red secretion60 SPF level NIH mice,18～22 g, male and female half and half. adapted to the environment for three days.And then divided randomly into five groups, control group, Vitex oil group, HaoHongfang high, medium and low dose group. The treatment group in accordance with the dose administered,20 ml/kg, such as dosage control group irrigated sesame oil.Once a day,7 days in all.1 h after the last administration,5% phenol red solution were injected with 0.01 ml/g intraperitoneal,0.5 h later, killed the mice by cervical dislocation, Yang-bit fixed-board cut in surgery cervical skin, separation of the trachea, tracheal strip surrounding tissue, cut the lower edge of thyroid cartilage to the tracheal bifurcation of the trachea, with 5% sodium bicarbonate solution,0.5 ml tracheal wash for 4 times, washing the combined solution, placed in UV-visible spectrophotometer on the wavelength of 546 nm measured in OD values, and in accordance with the standards set before the experiment to calculate tracheal fluid curve of phenol red were calculated expectorant rate.The formulation of the standard curve:analytical balance accurately weighed with a certain amount of phenol red to 5g/dl sodium bicarbonate dissolved in 1 ml phenol red to 1000 ug, followed by diluted per milliliter of phenol red 0.01,0.1,0.5,0.7,1,3,5 ug, test OD values to a dose of phenol red horizontal, OD value of the vertical axis for the standard curve, and calculated for each mouse phenol red secretion. There are differences between each group.2.3.6 HaoHongfang on the tracheal cilia movement of pigeonsPigeons,400～500 g, male and female in half, were randomly divided into 5 groups, namely control group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,10 ml/kg, once a day for 7 days, the control group was given the sesame oil.1 h after the last administration, straightened and paralleled levelly to the neck in pigeons in the dark room, and then stripped the trachea with a neck incision so as to expose the trachea, through the heart from the end of the needle NO.5 into the trachea, so that the needle close to the tracheal wall into the 0.02 ml ink, in the cold light source, the observed within 1 min from the forward movement of ink.2.4 The study of HaoHongfang on anti-inflammatory analgesic2.4.1 Inhibiting the capillary permeability in mice60 SPF level NIH mice, male,18-22g, adapted to the environment for three days, were randomly divided into 6 groups,10 each group, named control group, Aspen aspirin group, Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. In addition to the rest of control group were administered a dose of 20 ml/kg, once a days,7 days in all; 1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/lOg, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.2.4.2 The influence of HaoHongfang rat paw swelling of egg white60 SD rats,130～150 g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low groups 10. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil. Once a day,7 days in all. Before the last administration, each rat in the right rear paw Department as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline. After the last administration,1 h, each rat in the right foot plantar fascia under the skin injection of fresh egg white 100% 0.1 mL/only measured after injection of inflammatory agent 1,2,3,4 h after the right paw volume and less inflammation in rats before the right foot base value, as the mice at different times of the foot swelling. Comparison of different time levels of foot swelling.2.4.3 The influence of HaoHongfang rat paw swelling of formaldehydeSD rats were taken 60,140～160g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low group. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil.1 time per day, continuous intragastric administration 7 d. Prior to administration, in the right rear paw of each rat Office as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline.After the first dose 1 h, each rat in the right foot plantar fascia under the skin of 1% formaldehyde saline solution injection of 0.1 mL/only. Day 2 to 6 days to delivery, and measured at 1 h after administration right foot plantar volume after day 1.1,2,3,4,5 days after drug results to calculate foot swelling. Comparison of different time levels of foot swelling.2.4.4 The influence of HaoHongfang on the impact of pain in mice induced by acetic acid (writhing)72 SPF level NIH mice, male and female in half,18-22g, were randomly divided into 6 groups of 12 were named control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, mice were injected 0.8% acetic acid saline solution 0.1mL/10g weight, observed within 20 min the number of writhing writhing response: abdominal sag, stretch limbs, buttocks elevation), compared differences between each group.2.4.5 The influence of HaoHongfang on pain in the foot induced by formalin injection72 SPF level NIH mice, male and female in half,18～22 g, were randomly divided into 6 groups of 12 rats were named control group, aspirin group Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, all of the mice left and a full plantar subcutaneous injection of 2.5% formaldehyde saline solution 0.05 ml/only the immediate time, records of mice within 5 min left foot licking accumulated time difference between each group.2.5 The study of HaoHongfang on the immune function2.5.1 The influence of HaoHongfang on immune organsSPF level obtained NIH mice,60, male and female in half, weighing 13～15g, experimental environment to adapt to three days. According to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose group,12 in each group. The treatment group according to dose administered orally,20 ml/kg, once a day,7 days in all. After the last administration,12 h, mice were killed dislocation of cervical vertebra, rapidly profile control thymus, spleen, weight, organ index calculation. Spleen (thymus) index= spleen (thymus) weight (mg)/ body weight (g). There are differences between each group.2.5.2 The influence of HaoHongfang carbon particles clearance function in mice60 SPF level NIH mice, male,18-22 g, were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,20 ml/kg, once a day,7 days in all, the control group were given equal volume of sesame oil. After the last administration, 1h, mouse tail vein injection of diluted ink 0.1 ml/lOg weight, at 30 s after injection of ink, and 6 min, from the orbital venous plexus of mouse blood 0.025 ml, immediately cast them into the 2 ml 0.1% sodium bicarbonate solution; taking 0.025 ml normal human blood soluble ink injection 2 ml 0.1% sodium bicarbonate solution, zeroing,675 nm was determined at OD values of phagocytic index calculated by the formula K value. There are differences between each group.2.5.3 The influence of HaoHongfang on serum hemolysin formation of antibodies60 SPF level NIH mice, male and female in half,18～22g, according to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose groups 12. Each treatment group were administered orally by dose,20 ml/kg, once a day,10 days in all, the control group were given equal volume of sesame oil. Article 6, each mouse was injected with 5% sheep red blood cells 0.4 ml/only (equivalent to 4×108 SRBC/only). After the last administration, 1 h, orbital blood serum hemolytic antibody. There are differences between each group.2.5.4 The influence of HaoHongfang the impact on delayed-type hypersensitivity in mice60 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 5 groups, control group, Gynostemma group, HaoHongfang high, medium and low dose group,12 in each. Abdominal skin hair removaled (1 cm2 or so), covered with 50μl 1% DNFB sensitization,and the next day for the same. Each treatment group were orally administered by 20 ml/kg, day once,and for 10 days for all, the control group were given equal volume of sesame oil.7 days after administration of 1% DNFB solution evenly applied 10μl mouse right ear (both sides) to attack,24 hours later killed all of the mice and cut the left and right ears with the 8mm hole puncher, weighed, calculate swelling (DTH).3% Results:3.1 Study of Compatibility Rationality3.1.1 Inhibiting the capillary permeability in mice compared with the control group, Compound groups 3 (Artemisia 200mg/kg+Folium 100mg/kg) can statistically reduce the capillary permeability in mice (P<0.01); Compound group 1 (Artemisia annua 100mg/kg+ Folium 200mg/kg) and Compound group 2 (Artemisia 150mg/kg+Folium 150mg/kg) also can statistically reduce the capillary permeability in mice (P<0.05).3.1.2 Reduce cough in guinea pigs Compared with the control group, Compound group 2,3 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.01); Compound group 1 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.05).3.2 The study of HaoHongfang on antibacterial and antiviral3.2.1 Reduce the mortality rate of mice infected with Streptococcus pneumoniaeCompared with the control group, there were significantly higher mortality in model group (P<0.01),and had no effect on HaoHongfang control group. With model group comparison, HaoHongfang high and middle dose group was significantly lower mortality rate in mice (P＜0.01).3.2.2 Reduce the mortality rate of mice infected with Staphylococcus aureusCompared with the control group, model group mice were significantly higher mortality rate(P<0.01), and had no effect on HaoHongfang control group; With model group comparison, HaoHongfang high dose group was significantly lower mortality rate in mice infected by Staphylococcus aureus(P<0.01),and HaoHongfang middle and low group could also reduce mortality rate but there was no statistically different (P>0.05).3.2.3 Reduce the infection of influenza virus in miceCompared with the control group, the mortality rate,the average survival times of model group were significantly shorter (both P<0.01), but the were no influence in HaoHongfang control group. HaoHongfang high and middle dose group could significantly reduced the mortality rate in mice infected with influenza virus, prolong the average survival days(both P<0.01),and so did the HaoHongfang low-dose group (P<0.05).3.2.4 Inhibitory Lung-index of mice infected with influenza virusCompared with the control group, model group could significantly increased lung-index (P＜0.01); Compared with model group, HaoHongfang high dose group could reduce Lung-index in mice infected with influenza virus (P＜0.01), so did the middle group(P<0.05).3.2.5 Reduce the death in mice induced by endotoxinCompared with the control group, model group significantly increased mortality (P<0.01); compared with model group, high HaoHongfang significantly reduced the death in mice (P<0.01), HaoHongfang middle and low dose group could also decrease the death in mice but there was not significant difference (P> 0.05).4,Conclusion:The results show that HaoHongfang has antibacterial, antiviral, anti-endotoxin, Clearing heat transforming phlegm and suppressing cough, anti-inflammatory and pain releasement and immune enhancement function; provides pharmacodynamic basis for HaoHongfang in expelling wind-evil and clearing away heat, detoxification, phlegm and suppressing cough.