Wen ZHONG, Zujie QIN, Jiangcun WEI,*, Zhen XIE, Xiumei MA, Xianyi SHI, Fengxian ZHAO, Qian HAN

1. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] The effects of Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb on serum endotoxin, NO and TNF-α levels were investigated. [Methods] Total 105 mice were randomly divided into Dachengqi decoction made from raw rhubarb groups (6, 10 g/kg), Dachengqi decoction made from vinegar-processed rhubarb groups (6, 10 g/kg), positive control group, blank control group and model group. After administration at a dose of 20 mL/kg, the levels of endotoxin, NO and TNF-α in serum were determined. The effects of Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb on serum endotoxin, NO and TNF-α levels in mice were compared. [Results] Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb both increased the NO level and reduced the endotoxin and TNF-α levels in serum of ABP mice. [Conclusions] Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb have different effects on endotoxin, NO and TNF-α contents. These changes correspond to the effects of Dachengqi Decoctions made from raw rhubarb and vinegar-processed rhubarb. The change in the content of rhubarb anthraquinones has a certain effect on the efficacy of Dachengqi decoction.

Key words Rhubarb, Dachengqi decoction, Endotoxin, NO, TNF-α

1 Introduction

Dachengqi decoction was first recorded inTreatiseonFebrileDiseasesby Zhang Zhongjing in the Han Dynasty. It is composed of rhubarb, Fructus Aurantii Immaturus, Officinal Magnolia Bark, and mirabilite. Dachengqi decoction is the representative prescription of purgation[1-2], with effect of purging accumulated sthenia-heat, and it is mainly used for treating Yangming fu-viscera excess and interior heat[3]. At present, it is mainly used for the treatment of diseases such as acute abdomen, intestinal obstruction and gastrointestinal dysfunction. In addition, the prescription also has a good efficacy in improving the awareness of cerebrovascular patients and the rescue of acute poisoning with organophosphorus pesticides[4-5]. Research has shown that Dachengqi decoction has pharmacological effects such as inducing diarrhea, reducing inflammatory cytokines, inhibiting serum endotoxin, fighting inflammation and improving body immunity[4]. Rhubarb is used as a monarch drug in Dachengqi decoction, and its safety is determined by the Chinese herbs that make up the compound. After the preparation is processed, its properties are changed, and the toxic and side effects are reduced[6].

After being processed by different methods, the chemical composition and efficacy of rhubarb will change greatly, and the opposite effect may even produce[7]. The current reports are focused on the change in the efficacy of rhubarb after its compatibility with other herbs[8-9], but the differences in the purging efficacy of precipitations composed of different processed products of rhubarb, as well as the common relationship reflected in different compatibility have not been clarified. In this study, the effects of Dachengqi decoctions made from different processed products of rhubarb on endotoxin and NO and their interference with inflammatory mediator TNF-α in ABP mice were investigated to explore the effects of Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb on the changes of serum endotoxin, NO and TNF-α levels.

2 Materials and methods

2.1 Materials

2.1.1Instruments and equipment. Full-wavelength microplate reader (20110793, BIOTEK-Epoch, USA); low-temperature centrifuge (Shanghai Anting Scientific Instrument Factory); metabolic cage (ZH-B6, Anhui Zhenghua Co., Ltd.); incubator (PYX-190H-B, Guangdong Keli Co., Ltd.); PRACTUM224-KN SOP electronic analytical balance[Sedoris Scientific Instruments (Beijing) Co., Ltd.]; centrifuge (41284881 , Thermo Fisher, Germany).

2.1.2Drugs and reagents. Chromogenic End-point Tachypleus Amebocyte Lysate (TAL) (batch No.160525, Xiamen Bioendo Technology Co., Ltd.); ampicillin capsule (batch No.20300, The United Laboratories Ltd.); nitric oxide (NO) assay kit (batch No.20170406, Nanjing Jiancheng Bioengineering Institute); inflammatory mediator tumor necrosis factor (TNF-α) (batch No.2017/06, Nanjing Jiancheng Bioengineering Institute). The water used was pure water.

2.1.3Medicinal herbs. The used medicinal herbs included rhubarb (batch No.160307001, Sichuan), Fructus Aurantii Immaturus (batch No.160101771, Jiangxi), Officinal Magnolia Bark (batch No.160507021, Shaanxi) and mirabilite (batch No.160307611, Qinghai). Raw rhubarb, Fructus Aurantii Immaturus, Officinal Magnolia Bark and mirabilite were purchased from Guangdong Kangmei Pharmaceutical Co., Ltd. After identification by Tang Chunli, the chief pharmacist of the Pharmaceutical Department of the First Affiliated Hospital of Guangxi University of Chinese Medicine, rhubarb was the dried roots and rhizomes ofRheumofficinaleBaill., Fructus Aurantii Immaturus was the dried young fruit ofCitrusaurantiumL., Officinal Magnolia Bark was the dried root bark and branch bark ofMagnoliaofficinalisRehd. et Wils., and mirabilite was sulfate mineral, mainly containing sodium sulfate decahydrate (Na2SO4·10H2O).

2.1.4Animals. Kunming mice of SPF grade, weighing (20±2) g, half male and half female, were all provided by the Experimental Animal Center of Guangxi Medical University (production license No.SCXK Gui 2014-0002).

2.2 Methods

2.2.1Extraction of experimental liquid. According to the prescription, accurate amounts of powder of Officinal Magnolia Bark (24 g), Fructus Aurantii Immaturus (12 g), rhubarb (12 g) and mirabilite (9 g) were weighed. Officinal Magnolia Bark and Fructus Aurantii Immaturus were added with 500 mL of pure water, soaked for 30 min, boiled slightly for 30 min and filtered through 500-mesh filter cloth. The residue obtained was added with 500 mL of pure water, soaked for 30 min, boiled slightly for 30 min and filtered. The filtrate of the two times was mixed together, added with rhubarb powder, soaked for 20 min, boiled slightly for 20 min and filtered through 500-mesh filter cloth. The residue obtained was added with 300 mL of pure water, boiled slightly for 20 min and filtered. The filtrate was mixed, added with mirabilite while it was hot, stirred to make the mirabilite completely dissolve, concentrated into liquid solution (0.3∶1) containing 0.3 g/mL of crude drug, and stored in the refrigerator at 2-8 ℃ for future use.

In the same way, the drug solution was concentrated according to the ratio of 0.5∶1 (containing 0.5 g/mL of crude drug), and placed in a refrigerator at 2-8 ℃ for later use.

2.2.2Establishment of ABP mouse model. With reference to literature[10]and combined with pre-experiment, 15 healthy mice were randomly selected as the normal control group, and each of them was injected intraperitoneally with 0.5 mL of sodium chloride. A certain amount (6 g) of mouse feces was sampled, added with 100 mL of normal saline, ground evenly and let stand for 30 min. The upper suspension was collected and injected to the mice intraperitoneally according to a dose of 0.5 mL/mouse. After 20 h of intraperitoneal injection, the mice began to exhibit symptoms such as thick secretion in corner of eye, purple lip, less movement, vertical hair, crouching, abdominal bloating, no appetite and extremely dry stool. After dissection of abdominal cavity, if it was observed that there was a small amount of turbid liquid in the abdominal cavity, most of the intestinal tube was swollen, the intestinal tube was congested, and part of the intestinal tube was spasmed, it indicated that the modeling was successful. In clinic, Yangming fu-viscera excess could appear, and it is necessary to treat with cold purgation method.

2.2.3Drug administration. A total of 105 healthy mice, weighing (20±2) g, half male and half female, were selected after 3 d of adaptive feeding. They were randomly and evenly divided into the following 7 groups: 6 and 10 g/kg of Dachengqi decoction made from raw rhubarb groups (R-DCQD), 6 and 10 g/kg of Dachengqi decoction made from vinegar-processed rhubarb groups (V-DCQD), blank control group, model group and positive control group (ampicillin, 20 mL/kg body weight, 60 mg/mL).

In the Dachengqi decoction administration groups, the mice were administered after modeled successfully, one time a day, for two consecutive days. In the positive control group, the mice were given with ampicillin (0.06 g/mL) at a dose of 20 mL/kg after successful modeling, one time a day, for two consecutive days. In the blank control group, the mice were administered with distilled water by gavage at a dose of 20 mL/kg 20 h after intraperitoneal injection of normal saline, one time a day, for two consecutive days. In the model group, the mice were given with distilled water by gavage according to a dose of 20 mL/kg after successful modeling, one time a day, for two consecutive days. After administration, the levels of endotoxin, NO and tumor necrosis factor (NTF-α) in serum of the mice were determined.

2.2.4Determination of serum endotoxin content. After 42 h of successful modeling, the eyeball blood of the mice was collected aseptically, transferred in pyrogen-free tubes containing anticoagulant matched with the TAL reagent, placed in ice water, covered, and centrifuged at 3 000 r/min for 10 min at low temperature. An appropriate amount of supernatant in each tube was collected and diluted two times with endotoxin detection water. An accurate volume (0.2 mL) of the diluent of each sample was mixed with 0.8 mL of sample treatment solution to prepare into ten-fold dilutions for further use. The ten-fold dilutions were placed in a thermostatic water bath at 70 ℃ for 10 min (the tubes were sealed to prevent water drops from flowing into the samples), cooled with an ice water bath, and centrifuged at 3 000 r/min for 10 min at normal temperature. The supernatant of each sample was collected for detection, and the detection was carried out according to the instruction manual of the TAL kit. Quantitative determination was performed with the TAL chromogenic endpoint assay, and the results were expressed as eu/mL.

A total of 105 healthy mice, weighing (20±2) g, half male and half female, were selected. They were grouped and administered intragastrically according to the description in Section2.2.3. The administration dose was 6 g/kg, 10 g/kg or 20 mL/kg. After 20 h of intraperitoneal injection with fecal fluid, the mice were administered intragastrically once a day for two consecutive days.

2.2.5Determination of nitric oxide (NO) content. This study adopted the nitrate reductase method, and the operation was strictly in accordance with the kit instructions. A total of 105 healthy mice, weighing (20±2) g, half male and half female, were grouped and administered according to the method in Section2.2.3. The administration dose was 6 g/kg, 10 g/kg or 20 mL/kg. After 20 h of intraperitoneal injection with fecal fluid, the mice were administered intragastrically once a day for two consecutive days.

2.2.6Inflammatory mediator tumor necrosis factor (TNF-α). The double antibody sandwich enzyme-linked immunosorbent assay was used, strictly following the instructions of the kit. A total of 105 healthy mice, weighing (20±2) g, half male and half female, were grouped and administered according to the method in Section2.2.3. The administration dose was 6 g/kg, 10 g/kg or 20 mL/kg. After 20 h of intraperitoneal injection with fecal fluid, the mice were administered intragastrically once a day for two consecutive days.

2.2.7Statistical processing. The experimental data are expressed as mean±standard deviation. SPSS 21.0 statistical software was used for data analysis. One-way analysis of variance was used for the comparison of the means of multiple samples in a completely random design. If the difference was significant, SNK-q test was used for multiple comparisons among the means of multiple samples. WhenP<0.05, the difference is considered to be significant.

3 Results and analysis

3.1 Preparation of endotoxin standard solution and drawing of standard curveThe preparation method for endotoxin standard solution is shown in Table 1, and the standard curve drawn is shown in Fig.1.

Table 1 Endotoxin (ET) standard curve solutions

Note: Negative control was endotoxin inspection water.

Fig.1 Endotoxin (ET) standard curve

3.2 Serum endotoxin contentAs shown in Table 2, compared with that of the blank control group, the serum endotoxin contents of the R-DCQD group (6 g/kg), V-DCQD groups (6 and 10 g/kg), model group and positive control group were significantly different (P<0.01). Compared with that of the model group, the serum endotoxin contents of the R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg) and blank control group differed significantly (P<0.01). There were significant differences in serum endotoxin content between blank control group and R-DCQD group (6 g/kg) (P<0.01), between R-DCQD groups (6 and 10 g/kg) and V-DCQD group (10 g/kg), model group, positive control group (P<0.01), and between R-DCQD groups (6 and 10 g/kg) and V-DCQD group (6 g/kg) (P<0.05). The serum endotoxin content of the positive control group differed significantly from those of the R-DCQD groups (6 and 10 g/kg), V-DCQD group (6 and 10 g/kg) and blank control group (P<0.05,P<0.01).

Table 2 Effects on levels of endotoxin, NO and TNF-α in peripheral serum of ABP mice n=15)

Note: Compared with blank control group,*P<0.05,**P<0.01; compared with model group,●P<0.05,●●P<0.01; compared with R-DCQD groups,△P<0.05,△△P<0.01; compared with positive control group,#P<0.05,##P<0.01.

3.3 Serum NO contentCompared with that of the blank control group, the serum NO levels of the R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), model group and positive control group were significantly different (P<0.01). Compared with that of the model group, the serum NO contents of the R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), blank control group and positive control group showed significant differences (P<0.01). There were significant differences in serum NO level between V-DCQD groups (6 and 10 g/kg), blank control group, model group, positive control group and R-DCQD groups (6 and 10 g/kg), and between R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), blank control group, model group and positive control group (P<0.01) (Table 2).

3.4 Serum inflammatory mediator tumor necrosis factor (TNF-α) levelThe serum TNF-α levels of the R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), model group and positive control group were significantly from that of the blank control group (P<0.01). The serum TNF-α levels of the R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), blank control group and positive control group differed significantly from that of the model group (P<0.01). There were significant differences in serum TNF-α level between blank control group, model group, positive control group and R-DCQD groups (P<0.01), and between R-DCQD groups (6 and 10 g/kg), V-DCQD groups (6 and 10 g/kg), blank control group, model group and positive control group (P<0.01) (Table 2).

4 Discussion

Related research[11]shows acute infectious diseases and acute abdomen are manifestations of endotoxin biological activity, or caused by its induced cytokines and other inflammatory mediators. Substantive-heat-type constipation is a kind of Yangming fu-viscera excess. One of the pathogenic factors of substantive-heat-type constipation is endotoxin (ET). The generation and release of tumor necrosis factor (TNF-α) is the response of the body to various exogenous or endogenous substances. Endotoxin is the substance released by TNF-α. Both endotoxin and TNF-α can cause harm to the body. The combination of the two can produce a synergistic effect[10]. TNF-α can enter the blood circulation through the role of endocrine, and can also affect the adjacent intestinal epithelial cells through paracrine, thereby producing a certain impact on the intestinal barrier function[12]. Nitric oxide (NO) has a wide range of biological activities, widely distributed in various tissues in the body. It is a new type of biological messenger molecule, and plays a key role in the pathogenesis of multiple organ failure syndrome (MODS) caused by the intestine[13]. Related research[14]shows that NO is the last common pathway of the inflammatory mediator network and an important mediator that leads to organ damage. Systemic inflammatory response syndrome (SIRS) caused by inflammatory mediators is the main mechanism of the occurrence of multiple organ dysfunction syndrome (MODS). The excessive production of NO stimulated by bacterial endotoxin and inflammatory cytokines is a key mediating mechanism in the pathogenesis of septic shock[10].

The results of this study show that after fecal peritonitis (ABP) mice of substantive heat type were administered with Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb by gavage, the levels of NO and TNF-α in the serum of the mice decreased significantly. The differences in the serum endotoxin, NO and TNF-α levels in mice may be related to different processed products of rhubarb and its compatible Fructus Aurantii Immaturus, Officinal Magnolia Bark and mirabilite. The molecular mechanism for the differences needs further research.