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1 Download free from Original Article Effective Components of Dried Ginger in Warming Lung to Reduce Watery Phlegm and In vivo Tissue Distribution Based on the Syndrome-Efficacy Biological Sample Analysis Method Ying Cui a, Zheng Wang a, Ling-Ling Li a, Rui-Jie Li a, Jing Feng a, Kai Ma b, Bei Pan a, Yuan-Fei Niu a, Xing-Han Guo a, Yuan-Yuan Li a a Henan University of Chinese Medicine, Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment and Chinese Medicine Development of Henan Province, Zhengzhou , China, b Henan Province Chinese Medical Research Institute, Zhengzhou , China Abstract Objective: This study aimed to investigate effective components of dried ginger (DG) in warming lung to reduce watery phlegm and in vivo tissue distribution on the syndrome of cold fluid retained in lung of rats with chronic obstructive pulmonary disease (COPD) by means of the syndrome efficacy biological sample analysis method and then to explore its meridian tropism. Methods: Wistar rats were given nasal drops of 200 μl lipopolysaccharide and smoke 30 min two times a day, then put the appropriate dose of ice water, and freeze for an hour to build model rats. On the 16 th day, the drug group was orally administered of DG (500 mg/ml) until the 30 th day. Blood samples and biological tissues were collected from the orbital venous plexus into heparinized hemostasis tubes at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min after the last administration. Using ultraviolet high performance liquid chromatography (Waters, USA) method, the effective components were tested, and DAS 3.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used to analyze the results. Results: The compounds of DG entering into blood were 6 gingerol, 6 shogaol, and 8 gingerol. Tissue distribution analysis indicates that three active ingredients are widely present in the lung, spleen, kidney, liver, heart, large intestine, stomach, small intestine, and other organs of rats with COPD. Conclusions: 6 gingerol, 6 shogaol, and 8 gingerol belong to effective components of DG in curing the syndrome of cold fluid retained in lung of rats with COPD and mainly distributed in organs including the spleen, stomach, lung, kidney, liver, and heart. Keywords: Dried ginger, effective components, meridian tropism, syndrome of cold fluid retained in lung, syndrome efficacy biological sample analysis, tissue distribution Introduction Chronic obstructive pulmonary disease (COPD) is a relatively common chronic respiratory disease with high morbidity, disability, and mortality. As the most important syndrome of this disease, cold drink accumulation of lung is its high syndrome type. Cold drink accumulation of lung syndrome is a disease that occurs mostly due to the deficiency of lung yang, feeling cold again, so that water is stopped in the lung. Generally, it is a disease of Yang deficiency and cold prosperity. [1] Clinical manifestations include chest arrest, cough and wheezing, or phlegm thinning or frothy appearance, pale and vain complexion, edema of lower extremities, cold limbs, and white, greasy, or slippery tongue coating. [2] The disease develops progressively and is one of the diseases with the highest mortality rate among chronic diseases. [3] According Quick Response Code: Access this article online Website: DOI: /wjtcm.wjtcm_16_18 to the World Health Organization, the mortality rate of this disease is high and increasing year by year. [4] Dried ginger (DG) is a dry rhizome of ginger Zingiber officinale Rose and is one of the most commonly used drugs in the clinic. According to the 2015 edition of the Chinese Address for correspondence: Prof. Cui Ying, Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment and Chinese Medicine Development of Henan Province, Henan University of Chinese Medicine, Zhengzhou , China. E mail: wjtcmoffice@vip.126.com This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution NonCommercial ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com 2018 World Journal of Traditional Chinese Medicine Published by Wolters Kluwer Medknow Received: , Accepted: How to cite this article: Cui Y, Wang Z, Li LL, Li RJ, Feng J, Ma K, et al. Effective components of dried ginger in warming lung to reduce watery phlegm and in vivo tissue distribution based on the syndrome efficacy biological sample analysis method. World J Tradit Chin Med 2018;4: World J Tradit Chin Med Volume 4 Issue 3 July-September

2 Pharmacopoeia, [5] it is hot and spicy. It has special affinity for spleen, stomach, kidney, heart, and lung. DG has the effect of dispersing cold in warm, dispersing Yang, promoting blood circulation, warming lung, and resolving retained fluid. Besides, dry rhizome of Z. officinale Rose has the functions of warming middle, warming the lung, relieving cough, resisting aging, detoxifying, and preventing dizziness. [6] It is commonly used in the treatment of middle cold syndrome, Yang deficiency syndrome, and cold drink cough and asthma. There are more studies on the warm and back Yang of the DG but less for the study of the warm lung chemical drink. For this reason, we carried out the experimental study on the function of the warm and pulmonary drink of the DG on the rat cold drink syndrome model of the rat. Based on the above understanding, the author established a systematic method of Syndrome effect analysis of biological samples combined with the study of the effective substances of Chinese medicine and carried out an experimental study on the function of warm and pulmonary drink of DG on the syndrome model of cold drink and lung syndrome in rats. [7,8] However, the material basis of DG warming the lung is not clear. To this end, we used the disease syndrome effect biological sample analysis method, [9] on the basis of the effective intervention of DG in rats cold drink syndrome model of lung syndrome, to carry out the experimental study on the correlation between the distribution of the efficacy material in the warm lung beverage and the distribution of the effect materials in the body and the return of DG. Methods Reagents DG, produced from Sichuan Province, was purchased from Henan Zhang Zhongjing large pharmacy (Anguo City gold Kangdi medicines Ltd. production, production date: August 18, Batch number: ). Prof. Chen Suiqing from the Henan University of Chinese Medicine identified the dried rhizomes of ginger Z. officinale Rose. Lipopolysaccharide (LPS) (Sigma L 2880), pure methanol chromatography, TEDTA, pure chromatography (USA), cigarette smoke flower, and flue cured tobacco were used in this study. The tar content is 11 mg, the nicotine content of smoke is 0.7 mg, and the carbon monoxide of flue gas is 13 mg (Henan China Tobacco Industry Co., Ltd.). 6 gingerol, 6 shogaol, and 8 gingerol were all provided by the Henan Academy of Traditional Chinese Medicine (purity is >98%). Animals SPF class Wistar rat, male, weight (200 ± 20 g), purchased from Beijing Vitamin D Laboratory Animal Technology Co., Ltd., license number: SCXK (Jing) ; cage reared (20 C ± 2 C) laboratory, special people feed, feed standard full price feed. Preparation of rats After 5 days of feeding the rats, they were weighed after 24 h of fasting. According to the weight, they were randomly divided into blank group and the model group. On the 1 st day and 14 th day of the experiment, LPS solution (1 mg/ml) 200 μl was dripped into each rats nasal cavity. From the 2 nd to 30 th day of the experiment (except 14 th day). Every day at 8:30 a.m., the model rats were placed in the poisoned box (1 cigarette per mouse) to give smoke 30 min [9] and then filled with ice water 1 ml/100 g. The model rats were then placed in the environment of about 0 C for an hour, and at 2:30 p.m., the above operation was repeated in addition to the ice water. On the 16 th day of modeling, the model group was given the corresponding DG water decoction (500 mg/kg) until the end of the modeling. The blank group was given the same dose of DG water decoction. Collection and treatment of biological samples After the last irrigation of high dose group, two rats were taken at the same time for 5, 10, 20, 30, 40, 60, 90, 120, 240, and 360 min to drawn blood from eyeball, then killed them and took the lungs, heart, spleen, kidney, stomach, large intestine, small intestine, brain, bladder, and other tissues, and removed quickly. The animal blood was centrifuged at 8000 rpm for 10 min. One milliliter serum is injected in the 10 ml centrifuge tube, to this 2 ml methanol is added and the centrifuge tube is shacked for 5 min. The supernatant is placed in another 10 ml centrifuge tube and centrifuged at 8000 rpm for 10 min, and 2 ml methanol is added to the precipitate by combining the two supernatants. Nitrogen is dried at 40 C, to this add 2 ml of methanol, shake the solution for 5 min, and take the supernatant 1.5 ml over 0.22 μm microporous filter membrane, as the test solution. Clean the animal organs, dry the filter paper, weigh the animal tissue about 1 g, cut and set up the 10 ml centrifuge tube, add two times the amount of physiological saline (according to the proportion of sodium chloride injection = 1:2), and use the ultrasonic cell powder to homogenate the slurry. Accurately add two times methanol of the proportion of tissue, and vortex 5 min, then centrifuged at 8000 rpm for 10 min. The above operations are repeated two times. The nitrogen was dried at 40 C and then added to 2 ml of methanol. Shaked 5 min to fully activated and taked supernatant 1.5 ml to 0.22 μm microporous filter membrane as the test solution. Method for the determination of high performance liquid chromatography ultraviolet Chromatographic conditions Dikma Platisil ODS C 18 column (5 μm, 250 mm 4.6 mm), mobile phase: A is water, B is methanol, flow rate: 1.0 ml/min; detection wavelength: 227 nm; the gradient of elution is 60% B (0 min) 60% B (20 min) 70% B (30 min) 80% B (45 min) 100% (50 min) 60% B (55 min) 60% B (60 min). Column temperature: 30 C, sample 20 μl. 106 World J Tradit Chin Med Volume 4 Issue 3 July-September 2018

3 Preparation of mixed control solution Precise weighing standard substances, which included 8-gingerol 0.22 mg, 6-shogaol 0.1 mg, and 6-gingerol 1.22 mg in the same 10 ml volumetric flask, and fixed in methanol; the mixture of 8-gingerol, 6-shogaol, and 6-gingerol into mg/ml, 0.01 mg/ml, and mg/ml was prepared. Diluted to different concentrations, 8-gingerol into 22, 11, 5.5, 2.75, 1.375, , , , , and μg/ml, 6-shogaol into 10, 5, 2.5, 1.25, 0.625, , , , , and μg/ml, and 6-gingerol into 122, 61, 30.5, 15.25, 7.625, 3.813, 1.906, , , and μg/ml, respectively. Avoid light and reserved in 4 C environment. Specificity investigation The serum of the blank group and the homogenate of lung, large intestine, small intestine, brain, heart, liver, spleen, stomach, and kidney were 20 μl in accordance with the collection and treatment of 2.4 biological samples, and the samples were obtained to get the chromatogram of the blank sera and tissue samples. Sampling 20 μl of mixed standard solution and the mixed chromatogram was obtained. The mixed standard solution was added to the blank sera to be treated in accordance with the 2.4 biological sample collection, and the sample was injected into the sample of 20 μl, and the blank serum and tissue chromatograms containing the mixed standard were obtained. The comparison of the blank sera and tissue with the mixed standard solution and the comparison between the blank sera and the tissue chromatogram showed that the chromatographic conditions adopted in this experiment were suitable, the extraction method was appropriate, and the endogenous substances in the serum and tissue samples were not produced for the determination of 8 gingerol, 6 shogaol, and 6 gingerol. This method is highly exclusive. The retention time of 6 gingerol, 6 shogaol, and 8 gingerol are , , and min, respectively. Linear relation Precise absorption of mixed reference solution was 4, 6, 8, 10, 12, 14, 16, 18, and 20 μl. The standard curve was drawn with reference content as abscissa and peak area as ordinate. The results showed that 6 gingerol had a good linear relationship within mg range, the regression equation is Y = x , r = ; 6 shogaol has a good linear relationship in the range of mg, the regression equation is Y = x , r = ; and 8 gingerol has a good linear relationship in the range of mg, the regression equation is Y = 52936x , r = Precision The mixed reference substance was injected 20 μl into the liquid chromatograph, six times continuously, and the peak area integral value was determined. The relative standard deviation values of 8 gingerol, 6 shogaol, and 6 gingerol were 1.97%, 1.91%, and 1.94%, respectively, indicating that the precision of the instrument was good, as shown in Table 1. Accuracy The mixed standard solution, 200 μl (6 parts), was placed in the centrifuge tube, the nitrogen gas was dried at 37 C, and then, the lung homogenate was added to 200 μl, respectively. According to the collection and treatment of the samples of 2.3 tissues, the samples were mixed into a certain mixture of standard samples and tissues. After treatment, the samples were treated with 20 μl, respectively, analysis, the drug content was obtained by the obtained peak area, and the regression equation obtained before. The accuracy of three kinds of control samples obtained by this method was between 88% and 99%, which accorded with the quality control requirements of the biological sample analysis, and the specific results were shown in Table 2. Sample recovery In the centrifuge tube, 200 μl (6 parts) of the mixed standard solution was taken, and the nitrogen gas was dried at 37 C and then added to the lung homogenate 200 μl. The samples were mixed into the samples of the mixed standard samples and tissue in the centrifuge tube. After treatment, the samples were injected into the sample of 20 μl, respectively, and the peak area was recorded. Moreover, then the content of the chromatographic peak area of the backward sample treated with the blank biological sample is measured, and the recovery rate is calculated by the ratio of the actual mixed standard. The results are shown in Tables 3 5. The results showed that the extraction recovery rate of three kinds of control products in each tissue was between 59% and 73%, which was in line with the quality control requirements of biological sample analysis. Stability The mixed standard solution (6 parts) was taken in a centrifuge tube, and the nitrogen gas was dried at 37 C, and then, the blank sera was added to the 2.2 serum samples treatment method and the homogenate liquid of each tissue 200 μl Table 1: Test results of fine density Sampling times RSD (%) Peak area of 6 gingerol 2,990,679 2,993,742 2,985,096 2,989,893 2,993,897 2,990, Peak area of 6 shogaol 1,149,798 1,148,987 1,148,447 1,149,799 1,150,643 1,151, Peak area of 8 gingerol 520, , , , , , World J Tradit Chin Med Volume 4 Issue 3 July-September

4 according to the 2.3 tissue sample collection into the plug centrifuge tube and mixed into a certain concentration of the mixed standard samples and tissue samples. After treatment, according to the above chromatographic conditions, the sample was 20 μl, and a group of peak areas was obtained. The stability of the samples under room temperature of 24 h and 20 C was three times. The results of all the samples were <10%, and the stability accorded with the quality control requirements of the biological samples, as a result in Tables 6 and 7. Table 2: The accuracy of three kinds of reference substances in rat tissues was determined by high performance liquid chromatography Name of the sample Lung tissue 1 Lung tissue 2 Lung tissue 3 Lung tissue 4 Lung tissue 5 Lung tissue 6 Accuracy (%) 6 gingerol 2,843,789 2,896,782 2,990,676 2,874,654 2,765,087 2,788, shogaol 1,129,896 1,135,789 1,124,543 1,147,907 1,076,753 1,100, gingerol 508, , , , , , Table 3: 8 gingerol of the sample recovery rate of determination Number Sample quantity (µl) Addition of 8 Zingiber Real measurement Recovery rate (%) Average value (%) Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue RSD (%) Table 4: 6 shogaol sample recovery rate determination result Number Sample quantity (µl) Addition of 6 zingienol Real measurement Recovery rate (%) Average value (%) Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue RSD (%) Table 5: 6 gingerol sample recovery rate of determination Number Sample quantity (µl) Addition of 6 gingerol Real measurement Recovery rate (%) Average value (%) Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue Lung tissue RSD (%) Table 6: Stability test results for 24 h determination Name of the sample Lung tissue 1 Lung tissue 2 Lung tissue 3 Lung tissue 4 Lung tissue 5 Lung tissue 6 RSD (%) 6 gingerol 2,893,467 2,992,086 2,991,697 2,874,113 2,765,012 2,789, shogaol 1,129,865 1,136,544 1,124,010 1,147,832 1,078,047 1,107, gingerol 508, , , , , , World J Tradit Chin Med Volume 4 Issue 3 July-September 2018

5 Results High performance liquid chromatography spectrogram of mixed standard, medicinal material, serum with medicine, and blank serum After the analysis of the above blank serum, the serum of the drug, and the drug, it was found that 6 gingerol, 6 shogaol, and 8 gingerol were detected in the serum of the chronic obstructive pulmonary rats. It was shown that 6 gingerol, 6 shogaol, and 8 gingerol can be absorbed into the blood circulation. In the brain, heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, bladder, and other organs, 6 gingerol, 6 shogaol, and 8 gingerol were detected in different levels. It can be considered that the above three components of DG are related to the efficacy of treating COPD, as shown in Figures 1 4. Tissue distribution The results of the tissue distribution test of three components of 6 gingerol, 6 shogaol, and 8 gingerol in chronic obstructive pulmonary rats showed that in the organs such as brain, heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, and bladder, three effective ingredients of 6 gingerol, 6 shogaol, and 8 gingerol were detected. Analysis of pharmacokinetic parameters The obtained tissue distribution results were treated with DAS 3.0 to obtain the pharmacokinetic parameters of three active ingredients of 6 gingerol, 6 shogaol, and 8 gingerol in different tissues of chronic obstructive pulmonary rats. The AUC 0 t levels of 6 gingerol in tissues of chronic obstructive pulmonary rats were as follows: stomach > spleen > small intestine > lung > large intestine > liver > kidney > heart > brain > serum, 6 shogaol in cold dwelling lung tissue AUC 0 t followed by gastric > large intestine > lung > liver > small intestine. The Figure 1: High performance liquid chromatography spectrogram of mixed standard Figure 2: High performance liquid chromatography spectrogram of dried ginger Figure 3: High performance liquid chromatography spectrogram of serum with medicine Figure 4: High performance liquid chromatography spectrogram of blank serum Table 7: Test results of repeated freezing and thawing three times for the stability test Name of the sample Lung tissue 1 Lung tissue 2 Lung tissue 3 Lung tissue 4 Lung tissue 5 Lung tissue 6 RSD (%) 6 gingerol 2,913,469 2,899,762 2,991,666 2,874,206 2,765,107 2,788, shogaol 1,129,797 1,136,543 1,123,892 1,147,806 1,078,179 1,107, gingerol 508, , , , , , World J Tradit Chin Med Volume 4 Issue 3 July-September

6 AUC 0 t of 8 gingerol in the cold intake lung tissue was gastric > large intestine > spleen > small intestine > serum > liver > brain > lung. The specific results are shown in Tables Discussion Ginger efficacy related substances DG has effect in warming lung and relieves cough and asthma. Therefore, it is necessary to observe the efficacy of DG and warm lung in the model of chronic obstructive pulmonary rats. The tissue distribution data of DG in chronic obstructive pulmonary rats model showed that the contents of 6 gingerol, 6 shogaol, and 8 gingerol in DG were found in blood, brain, heart, liver, spleen, lung, kidney, stomach, and bladder; large intestine and the small intestine were detected. Therefore, it can be considered that the gingerol components represented by 6 gingerol, 6 shogaol, and 8 gingerol contained in DG are the related functional substances for the treatment of chronic obstructive pulmonary rats with DG. The correlation between tissue distribution of efficacy related substances in dried ginger and meridian channel tropism AUC 0 t (AUC 0 t refers to the total area under the drug time curve for the drug from time zero to all the drugs in the original form, reflecting the total amount of drug entering the blood circulation), the earliest inspection time, maintaining time, and other information can objectively reflect the distribution of drugs into the body. In the chronic obstructive pulmonary rat model, the order of the first detected in the 6 gingerol tissues in DG is as follows: spleen = stomach = large intestine = serum = small intestine > Table 8: Pharmacokinetic parameters of ginger 6 gingerol in ginger extract from rats in the chronic obstructive pulmonary rat group Tissue AUC 0 t (mg/l*min) t 1/2 (min) T max (min) C max (mg/l) CL/F (L/min/kg) Serum Heart Liver Spleen Lung Kidney Stomach Brain Large intestine Small intestine AUC: Area under the curve, CL/F: Scavenging rate Table 9: Pharmacokinetic parameters of ginger 6 shogaol in ginger extract from rats in the chronic obstructive pulmonary rat group Tissue AUC 0 t (mg/l*min) t 1/2 (min) T max (min) C max (mg/l) CL/F (L/min/kg) Liver Lung Stomach Large intestine Small intestine AUC: Area under the curve, CL/F: Scavenging rate Table 10: Pharmacokinetic parameters of ginger 8 gingerol in ginger extract from rats in the chronic obstructive pulmonary rat group Tissue AUC 0 t (µg/l*h) MRT 0 t (h) t 1/2 (h) T max (h) C max (µg/l) CL/F (L/h/kg) Serum Liver Spleen Lung Large intestine Small intestine Brain Stomach MRT: Mean residence time, AUC: Area under the curve, CL/F: Scavenging rate 110 World J Tradit Chin Med Volume 4 Issue 3 July-September 2018

7 lung = bladder > kidney > liver = heart > brain, the order of AUC 0 t in each tissue is as follows: stomach > spleen > small intestine > lung > colon > liver > kidney > heart > brain > serum, and the order of time maintained in each organization is as follows: spleen > stomach > lung = small intestine > colon > bladder > heart > kidney > liver > serum = brain. In the chronic obstructive pulmonary rat model, the order of the first detected in the 6 shogaol tissues in DG is as follows: serum = small intestine = stomach = lung = spleen > large intestine > liver > brain > bladder > kidney > heart, the order of AUC 0 t in each tissue is as follows: stomach > large intestine > lung > spleen > liver, and the order of time maintained in each organization is as follows: stomach > lung > spleen > liver = large intestine > lung = bladder = heart = kidney > brain > serum. In the chronic obstructive pulmonary rat model, the order of the first detected in the 8 gingerol tissues in DG is as follows: serum = small intestine = lung = stomach > spleen = brain > large intestine = kidney = liver = bladder > heart, the order of AUC 0 t in each tissue is as follows: stomach > kidney > spleen, and the order of time maintained in each organization is as follows: stomach > spleen = small intestine > lung > large intestine > brain = liver > serum > kidney > heart > bladder. The above experimental data showed that the three components of DG were absorbed most rapidly by spleen, small intestine, and stomach, which lasted for a long time with great intensity and in the literature have documented ginger treatment of spleen deficiency syndrome in spleen stomach. The three components of DG were detected early in the lung tissue, of which 6 gingerol and 6 shogaol AUC 0 t were large, and the absorption rate was very fast, and the time of maintenance was very long. This provides a basis for drug distribution of the DG belonging to the lung channel and cure chronic obstructive pulmonary. In summary, the results of this experiment show that the distribution of the representative components of DG is consistent with the Chinese traditional understanding of the DG belonging to the lung and spleen and stomach channel. Conclusions The composition of DG into the body was analyzed under the condition that the DG interferes with chronic obstructive pulmonary rat model. 6 gingerol, 6 shogaol, and 8 gingerol in DG are the functional substances of DG. The distribution of 6 gingerol, 6 shogaol, and 8 gingerol in the model is consistent with the traditional understanding of DG belonging to the spleen and stomach and lung Meridian. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. References 1. Lu SY. The Dynamic Expression of AQP1, Muc5ac in Chronic Obstructive Pulmonary Disease Model Rats of Syndrome of Accumulation of Cold Fluid in the Lung in Lung Tissue. Traditional Chinese Medicine of Shandong University; Wang HW. Establishments of the Rat Model of COPD Syndrome of Cold rheum Lying Latent in the Lung. Traditional Chinese Medicine of Shandong University; Wang Q. Progress in the treatment of chronic obstructive pulmonary disease in stable phase [J]. Chinese modern doctor, 2009, 47(30): Wang HY, Wang S. Research progress of Chinese medicine in treating chronic obstructive pulmonary disease [J]. Clinical Journal of Traditional Chinese Medicine 2018; 30(02): Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia, Part 1. Beijing: China Medical Science and Technology Society; p Zhang JX, Li LF. Pharmacological action of ginger. Hebei Med 1993;15: Cui Y, Yang JJ, Guo MJ, Wang JM, Zhao SX, Ma Kai, et al. Investigation on An-shen drug substances and meridian tropism of semen zizyphi spinosae based on syndrome-effect-analysis of biological samples. World Sci Technol Modernization Chin Med 2015; 17: Cui Y, Ji B, Feng ZY, Feng J. Studies on the efficacy of ginger in Chinese medicine. Chin Med Clin 2011;2:27-9.e Sun GR, Wang HW, Gao B, Xue WG. COPD Hanyinyunfei syndrome rat model of the establishment of the disease. J Shandong Univ Tradit Chin Med 2007;31: World J Tradit Chin Med Volume 4 Issue 3 July-September