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EXPERIMENTAL RESEARCH
Differences in Effects of Zuojin Pills () and Its Similar Formulas on Wei Cold Model in Rats
ZHAO Yan-ling ()1, SHI Wen-li ()2, SHAN Li-mei ()1, WANG Jia-bo ()1, ZHAO Hai-ping ()1, and XIAO Xiao-he ()1
ABSTRACT Objective: To explore the effects of Zuojin Pills () and its similar formulas on the stomach

cold syndrome in a Wei cold model in rats. Methods: The rat Wei cold model was established by intragastric administration of glacial NaOH, and the gastric mucosa injury indices, together with the levels of motilin and gastrin in the stomach, were determined. The preventive and curative effects of Zuojin Pills and its similar formulas on gastric mucosa injury were investigated. Results: Zuojin Pills and its similar formulas could protect the gastric mucosa in the gastric cold model in rats at different levels. Fanzuojin Pills () had the best effect in inhibiting gastric mucosa injury. Conclusion: The different pharmacological effects of Zuojin Pills and its similar formulas in the rat gastric cold model were partially correlated with the degrees in cold and heat properties of the formulas. KEY WORDS Zuojin Pills, similar formula, rat, Wei cold syndrome

The formation of famous classical formulas in Chinese medicine is ingenious. Moreover, the similar formulas in Chinese medicine are similar in appearance but different in nature. The four similar formulas recorded in Dan Xi Xin Fa (), Sheng Ji Zong Lu (), and Tai Ping Sheng Hui Fang Zhi Shui Xie Zhu Fang ( ) consist of Rhizoma Coptidis and Fructus Evodiae . However, the pharmacological effects are different because of different ratios between Rhizoma Coptidis and Fructus Evodiae (1,2). Zuojin Pills (), which is made of Rhizoma Coptidis and Fructus Evodiae with a ratio of 6:1, has been good for the treatment of Wei () heat syndrome because of the large ratio of Rhizoma Coptidis with cold property. Fanzuojin Pills (), which consists of Rhizoma Coptidis and Fructus Evodiae with a ratio of 1:6, has been good in treating Wei cold syndrome because of the large ratio of Fructus Evodiae with hot property. Ganlu Powder () and Zhuyu Pills () consisting of Rhizoma Coptidis and Fructus Evodiae with ratios of 2:1 and 1:1, respectively, have been used in the treatment of summer qi diseases and deficient cold dysentery, respectively, because of the ratio of cold and hot herbs located in the middle of Zuojin Pills and Fanzuojin Pills. In this research, as the rat Wei cold model was used as an objective index, the correlation between the ratio of cold and hot herbs and the effects on Wei cold syndrome were analyzed. Also, the characteristics and differences of the similar formulas

in Chinese medicine were analyzed.

METHODS
Drugs
(1) Zuojin Pills (the ratio of Rhizoma Coptidis : Fructus Evodiae =6:1, 3.45 g crude herb per gram dry paste powder); (2) Fanzuojin Pills (Rhizoma Coptidis :Fructus Evodiae=1:6, 2.69 g crude herb per gram); (3) Ganlu Powder (Rhizoma Coptidis : Fructus Evodiae =2:1, 2.99 g crude herb per gram); and (4) Zhuyu Pills (Rhizoma Coptidis: Fructus Evodiae=1:1, 2.93 g crude herb per gram). The dosage for rats was 7.2 g crude herb/kg prepared using distilled water, which was equivalent to 2 g dried paste powder of Zuojin Pills, 2.6 g Fanzuojin Pills, 2.4 g Ganlusan, and 2.5 g Zhuyu Pills, respectively.

Reagents
Sodium hydroxide, Beijing Chemical Reagent Company, Lot number: 060109. Both gastrin (GAS) and motilin (MTL) Kits were provided by the
Supported by National Basic Research Program of China (No. 2007CB512607), National Nature Science Foundation of China (No. 30772740) 1. Institute of Traditional Chinese Material Medica, the 302 Hospital of PLA, Beijing (100039), China; 2. Department of Integrated Traditional and Western Medicine, the 302 Hospital of PLA, Beijing (100039), China Correspondence to: Prof. XIAO Xiao-he, Tel: 86-10-66933322, E-mail: pharmacy302@126.com DOI: 10.1007/s11655-009-0293-7

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Chin J Integr Med 2009 Aug;15(4):293-298 2.93 g of Zhuyu Pills dried powder were administered orally with the dosing volume of 10 mL/kg. The effect of the drugs on the injury indices was observed after drug treatment.

Beijing Beifang Biology Institute, China. Superoxide dismutase (SOD, Lot number: 20070823) and malondialdehyde (MDA, Lot number: 20070508) were purchased from the Nanjing Jiancheng Biological Engineering Institute, China.

Determination of Serum SOD and MDA Animals

Sprague-Dawley (SD) rats weighing 180200 g were obtained from Beijing Vital River Experimental Animal Technology Co., Ltd., certificate No. SCXK (Jing) 2007-2010. All animals were kept under the same laboratory conditions at 25-27 in the 12:12 h light: dark cycle, with free access to standard laboratory chow and tap water. Blood samples were collected from the rat femoral artery. The contents of SOD and MDA were determined according to the protocol with the commercial kits.

Determination of GAS and MLT of Blood and Gastric Mucosa

Blood samples were collected from the rat femoral artery. Then, the stomach was removed from the animal, and the gastric mucosa was isolated. The contents of GAS and MTL were determined according to the protocol with the commercial kits.

Apparatus
DM-1 anatomy microscope was provided by Xiamen Shennuo Equipment Co., Ltd., China DDL low-temperature centrifuge was purchased from the Shanghai Anting Science Instrument Factory, China.

Injury Index Determination

In the cases of mucous membrane injury, the length was examined, and the score of one point was calculated for one millimeter injury. If the length was over one millimeter, the score was doubled. For punctiform hemorrhage (length and width less than 1 mm), the score of 0.5 was calculated. The area was examined when hemorrhage was at the state of plaque (both length and width over 2 mm). All above examined results added were the injury index of the rat (4,5). The ratio of injury inhibition was calculated as follows. Ratio of injury inhibition=(Average injury index of model group-Average injury index of drug treatment group)/Average injury index of model group 100%.

Gastric Mucosa Injury Model with Wei Cold Syndrome

According to the method(3), rats were given free access to standard laboratory chow and tap water on the first day of experiment, while 4 glacial water was administered orally at the dose of 20 mL/kg three times per day at 6-h intervals. On the second day, the rats were made to fast, but free to tap water for 24 h and the glacial water was administered as mentioned above. On the third day, the rats were administered with 4 0.3 mol/L glacial NaOH at the dose of 10 mL/kg to prepare the gastric cold model. The animals were sacrificed 24 h later, and the stomach was collected; 10% formaldehyde was perfused into the stomach for fixation; 10 min later, the stomach was torn apart along the gastric greater curvature and was washed to clean. The length of injury was examined under the anatomical microscope.

Statistical Analysis
All injury indices were expressed as mean standard deviation. Differences between group means were calculated by one-way analysis of variance of SPSS 12.0 for windows. A value of P <0.05 was considered statistically significant.

Experimental Protocol
The protocol of pretreatment and remedial treatment was adopted, and the rats were randomly divided into six groups of ten rats each, including normal group, model group, Zuojin Pills group, Fanzuojin Pills group, Ganlusan group, and Zhuyu Pills group. The drug was administered once a day for six days starting three days before model making. Two gram of Zuojin Pills dried powder, 2.69 g of Fanzuojin dried powder, 2.99 g of Ganlusan dried powder, and

RESULTS
Effects of Zuojin Pills, Fanzuojin Pills, Ganlusan, and Zhuyu Pills on Mucosa Injury in Wei Cold Model
In the model group, separate red and white or red and purple red injury was noted in the gastric mucosa, together with blood vessel stripes transparently seen when examined by macroscopic

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D
Figure 1.

Protective Effects of Zuojin Pills and Its Similar Formulas on Gastric Mucosa of Wei Cold Syndrome Model in Rats

Notes: A: normal group; B: model group; C: Zuojin Pills group; D: Fanzuojin Pills group; E: Zhuyu Pills group; F: Ganlu Powder group

observations. The mucosa showed rough appearance with obvious hyperemia, ulcer, and erosion injury at different levels. Compared with the model group, the injury has been ameliorated to some extent in the groups of drug treatment. The pharmacological effect of Fanzuojin Pills is equivalent to Zuojin Pills in the protection of the gastric mucosa. The macroscopic observation showed the smooth mucosa with incidental mottling injury. Analyzed by statistic software, the injury index of the gastric mucosa in drug treated groups was significantly different when compared with the model group (Table 1 and Figure 1). The single-factor multilevels(6) statistical analysis showed that when compared with Fanzuojin Pills and Ganlu Power, respectively, Zuojin Pills showed significant difference (P <0.01, P <0.05). There was no differences between Ganlu Power and Zhuyu Pills (P >0.05), while a significant difference could be seen between Ganlu Power and Fanzuojin Pills (P <0.01). It showed a significant difference when Zhuyu Pills was compared with Fanzuojin Pills (P <0.01).

Table 1. Effect of Zuojin Pills and Its Similar Formulas on Gastric Mucosa Injury in Gastric Cold Model Rats ( s )
Group Model Zuojin Pills Fanzuojin Pills Ganlusan Zhuyu Pills 2 2.69 2.99 2.93 Dose (g/kg)

Injury index

Inhibitory ratio (%) 76.21 86.24 66.56 71.89

10 118.2617.38 10 10 10 10 28.136.72 16.275.82 39.557.64 33.246.87

Note: P <0.05, P <0.01, compared with the model group

200 150 SOD (U/mL) 100 50 0 Normal Model Zuojin Pills Fanzuojin Pills Ganlu Powder Zhuyu Pills

Groups

Figure 2. Effects of Zuojin Pills and Its Similar Formulae on Level of SOD in Gastric Cold Syndrome Rats

Effect of Zuojin Pills and Its Similar Formulas on Serum SOD and MDA
Having collected blood from the rat femoral artery, the content of SOD and MDA were determined according to the protocol accompanied with the commercial kits(7). Compared with the normal control, the activity of SOD was decreased, while the level of MDA elevated in the model rats. Zuojin Pills could increase the SOD value from 118.3716.54 U/mL (model group) to 131.6721.28 U/mL, while the level

Notes: P<0.01, compared with the normal group; P<0.05, P<0.01, compared with the model group

of SOD in rats treated with Fanzuojin Pills was up to 138.9117.45 U/mL, Ganlu Powder reached 126.54 31.26 U/mL, and Zhuyu Pills reached 128.6926.68 U/ mL. Meanwhile, the serum MDA level in the model group rats was increased from 5.870.72 nmol/mL in normal rats to 9.120.46 nmol/mL. Zuojin Pills decreased the MDA level to 7.220.53 nmol/mL, while Fanzuojin, Ganlu Powder, and Zhuyu Pills reduced the MDA level to 7.810.64 nmol/mL, 7.460.36 nmol/mL, and 7.32

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Effect of Zuojin Pills and Its Similar Formulas on GAS and MTL in Gastric Cold Syndrome Rats ( s )
n
10 10 10 10 10 10 MTL (pg/mL) Blood 268.6719.62 209.1415.73 229.8216.33 238.7521.54 231.6818.34

Table 2.
Group Normal Model Zuojin Pills

GAS (pg/mL) Blood 42.338.96 28.159.87 34.548.46 36.425.35 31.554.52

Gastric mucosa 226.8319.86 186.4617.16 199.8834.58 206.5121.11 202.1518.23

Gastric mucosa 678.6918.38 493.4523.12 563.6817.83 579.8219.96 553.7726.98 555.8126.19

Fanzuojin Pills Ganlusan Zhuyu Pills

236.7822.56

204.1626.88

33.685.87

Notes: P <0.01, compared with the normal group; P <0.05, P <0.01, compared with the model group

0.48 nmol/mL, respectively (Figures 2 and 3).

Effect of Zuojin Pills on Levels of MTL and GAS in the Blood and Gastric Mucosa
Compared with the normal group, the contents of MTL and GAS in blood and gastric mucosa were significantly decreased in the model group rats (P <0.01). Zuojin Pills, Fanzuojin Pills, Ganlusan, and Zhuyu Pills increased the levels of MTL and GAS in blood and gastric mucosa compared with the model group (P <0.05, P <0.01, Table 2).
12 10 MDA (nmol/mL) 8 6 4 2 0 Normal Model Zuojin Pills Fanzuojin Pills Ganlu Powder Zhuyu Pills

Groups

Figure 3. Effects of Zuojin Pills and Its Similar Formulae on Level of MDA in Wei Cold Syndrome Rats
Notes: P<0.01, compared with the normal group; P<0.01, compared with the model group

DISCUSSION
Rhizoma Coptidis is bitter cold, while Fructus Evodiae is pungent warm in nature. When these two herbs with different properties are used together, the pungent drug is used for dispersion and the bitter drug for purgation, while yin and yang were in coordination. Since the indication of Zuojin Pills and its formulas were in the gastrointestinal tract, the gastric mucosa of rats was regarded as an objective index to observe the different effects among Zuojin Pills and its formulas on the injury of the gastric mucosa by establishing gastric mucosa injury model of Wei cold syndrome.

In clinics, bile regurgitation can induce injury on the gastric mucosa and result in acute or chronic reflux gastritis, which was mainly because of alkaline substances existed in the contraflow liquid (8). The pain induced by sodium hydroxide infusion into the stomach of the patients with bile reflux gastritis was similar to that of gastritis(9). Since this kind of pain is commonly seen in patients of Pi ( ) and Wei deficiency cold syndrome, NaOH was used to induce the gastric mucosa injury in the Wei cold syndrome(10). Meanwhile, it is considered theoretically in Chinese medicine that one of the causes for gastrointestinal diseases was adephagia in crude, cold, chilly, and cool food. That is also the reason why NaOH was selected to induce the injury of the gastric mucosa on Wei cold model in rats(11). From the observation on mucosa morphology, the color of the gastric surface was pale or gloomy, which was in accordance with the Chinese medicine theory of cold being in charge of coagulation and stagnant. According to the research results on relationship between gastric cold syndrome and gut hormones, the gut hormone changes play very important roles in the occurrence of the gastric cold syndrome. Part of the Wei cold syndrome was induced by endocrine functional disorders of the gastrointestinal system. Therefore, understanding the gut hormone changes such as Wei and notilin will be helpful to further explore the essence of Wei cold syndrome(12). In this experiment, the different protective effects of Zuojin Pills and its similar formulas with herbs of cold or hot properties in compatibility in different ratios were investigated in the gastric mucosa of the rat gastric cold model. Meanwhile, the different effects among Zuojin Pills and its formulas on Wei cold syndrome were analyzed. Researches showed that gastric mucosa injury

Chin J Integr Med 2009 Aug;15(4):293-298 was closely related with oxygen free radicals (13-15). Imbalance of oxygen free radical metabolism plays important roles in gastric mucosa injury, and lipid peroxidation is the main reason. It has been generally accepted that SOD activity decreases in the gastric mucosa tissue of patients with chronic gastritis (16). Oxygen free radicals can induce lipid peroxidation of the gastric mucosa or mucosa cells both in vivo and in vitro and cause gastric mucosa injury. SOD, as one of the most important marker enzymes in the defensive clearance system, is highly active in gastric parietal cells and can reflect the capability of the organism in free radical clearance. MDA is the final product of lipid peroxidation, and the detected MDA levels could be used to reflect the level of oxygen free radicals. The results in this experiment showed that the activity of SOD in the model rat blood was decreased, while MDA was elevated. Zuojin Pills, Fanzuojin Pills, Ganlu Powder, and Zhuyu Pills could elevate the SOD level and reduce the MDA level. The results indicated that Zuojin Pills and its similar formulas could remove oxygen free radicals from the gastric mucosa and could inhibit lipid peroxidation in serum by improving the activity of SOD to protect the gastric mucosa. Meanwhile, the reparation of acute mucosa injury could be promoted, and the ulcer healing could also be improved. In recent years, research indicated that endogenous MLT could be produced by gastric mucosa cells. GAS could protect the gastric mucosa in rats and promote the healing of gastric mucosa injury. Glacial water plus glacial NaOH could reduce the content of motilin and gastrin in the blood and gastric mucosa in rats, while Zuojin Pills, Fanzuojin Pills, Ganlu Powder, and Zhuyu Pills could increase the MTL and GAS in the blood and gastric mucosa from the level of the model group. By observation of gastric mucosa injury, the conclusion could be drawn that Fanzuojin Pills had the best effect in the gastric cold model, demonstrating that the formula was correspondent with syndrome. Although Zuojin Pills and its similar formulas all had protective effects on the Wei mucosa, different effects could be seen in different syndromes. On the other hand, the fact that Zuojin Pills and its similar formulas had protective effects on the gastric mucosa provided bases for further development of new drugs. In this experiment, the Wei cold syndrome model was mainly manifested as the cold syndrome. In this model, the pharmacological effect of Fanzuojin Pils was better

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than Zuojin Pills. According to the theory of treating the cold syndrome with hot-natured drugs while heat syndrome with drugs of cold or cool nature, hot-natured drugs produced marked effects in the cold model. In conclusion, among the formulas tested, Fanzuojin Pills could significantly improve the gastric mucosa injury in the rat Wei cold model with the best pharmacological effect, which was in accordance with formula explanation. Researches showed that the ratio of Rhizoma Coptidis and Fructus Evodiae in compatibility is related to the major compositions of the formula. In addition, the superior pharmacological effect of Zhuyu Pills over Ganlu Powder used in hotsyndrome clearance demonstrated its therapeutic effects on deficient cold dysentery.

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