Effects of Ethanol Extract of Laggera alata (D. Don) Sch. Bip. ex Oliv on Cirrhotic Rats with Ascites

Jiangcun WEI, Haisheng ZENG, Xiumei MA, Jingwen GU, Mengxia YANG, Qian HAN, Fengxian ZHAO

1. Guangxi International Zhuang Medicine Hospital, Nanning 530201, China; 2. First Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China; 3. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] This study aimed to investigate the intervention effects of ethanol extract of Laggera alata (D. Don) Sch. Bip. ex Oliv on cirrhotic rats with ascites. [Methods] A total of 100 male SD rats of SPF grade were used. Rat models of cirrhosis and ascites were established. After the last administration, the amount of peritoneal fluid was measured, and the blood of each rat tested was collected for determination of serum AST, ALT and ALB levels, serum K+ concentration and plasma Ald level. [Results] In ascitic cirrhotic rats, the livers showed obvious enlargement, the surface of the livers was uneven, and nodules were visible. Compared with the blank group, the amount of peritoneal fluid in the model group increased significantly (P<0.01); and compared with the model group, the amounts of peritoneal fluid in the high and medium-dose ethanol extract groups reduced significantly (P<0.01). Compared with the blank group, the serum ALT and AST levels in the model group increased significantly (P<0.01); and compared with the model group, the serum ALT and AST levels in the high-dose ethanol extract group decreased significantly (P<0.05). Compared with the blank group, the serum K+ concentration in the positive group reduced (P<0.05); no significant difference was found in serum K+ concentration between the ethanol extract administration groups and the blank group (P>0.05). Compared with the blank group, the plasma Ald levels in the high-dose ethanol extract group and the positive group reduced (P<0.05); and there was no significant difference in serum K+ concentration between the rest groups and the blank group (P>0.05). [Conclusions] In the ethanol extract administration groups, the amounts of peritoneal fluid significantly reduced, the serum ALT and AST reduced, and the liver functions improved to a certain extent, indicating that the ethanol extract of L. alata (D. Don) Sch. Bip. ex Oliv has a better correction effect on electrolyte disturbances in cirrhotic rats with ascites.

Key words Laggera alata (D. Don) Sch. Bip. ex Oliv, Cirrhosis with ascites, Extract

1 Introduction

Laggeraalata(D. Don) Sch. Bip. ex Oliv (Asteraceae:Laggera) is used as medicine with its whole plant. It is bitter and spicy in taste and slightly warm in nature, with effects of expelling wind, drying dampness and detoxifying. In the folk, it is a good medicine for anti-bacteria, anti-inflammation, clearing heat and detoxification[1].L.alata(D. Don) Sch. Bip. ex Oliv is commonly used for various diseases such as traumatic injuries, abdominal pain, diarrhea, cold, cough, snake bite and rheumatoid arthritis.L.alata(D. Don) Sch. Bip. ex Oliv contains chemical components such as sesquiterpenoids, flavonoids[2]and phenolic acids[3-6]. Related pharmacological experiments proveL.alata(D. Don) Sch. Bip. ex Oliv has the effects of treating acute and chronic inflammation, anti-tumor, anti-virus and anti-microbia[3,7-8]. The preliminary experiment[9]showed thatL.alata(D. Don) Sch. Bip. ex Oliv has good anti-inflammatory and anti-hepatitis B effects. In this study, the effects of extract ofL.alata(D. Don) Sch. Bip. ex Oliv on cirrhosis with ascites were investigated, and the mechanism of action was explored preliminarily, in order to provide an experimental basis for the further development and application of this medicinal plant.

2 Materials and methods

2.1 Materials

2.1.1Instruments and equipment. Full-wavelength multifunctional microplate reader (Infinite M200 PRO, Shanghai Anjing Technology Co., Ltd.); electronic balance[PL203, Mettler-Toledo Instruments (Shanghai) Co., Ltd.]; automatic biochemical analyzer (Shanghai Kehua Biological Engineering Co., Ltd.); ultra-low temperature freezer (DW-86L490, Qingdao Haier Co., Ltd.); ultra-thin slicer (Leica EM C7, Changzhou Philas Instrument Co., Ltd.); paraffin embedding machine (LeicaEG1150H+C, Changzhou Philas Instrument Co., Ltd.); automatic tissue dehydrator (Leica ASP300S, Changzhou Philas Instrument Co., Ltd.).

2.1.3Animal and experimental environment. A total of 100 male Sprague-Dawley (SD) rats of SPF (specific pathogen free) grade, weighing (180-220)g, were purchased from Experimental Animal Center of Guangxi Medical University[license number SCXK (Guangxi) 2014-0002]. The experimental environment was as follows: clean animal room, temperature of (25 ± 1) ℃, humidity of about 65%, artificial photoperiod (12-h light and 12-h dark). The feed used was standard rodent feed. The drinking water used was purified water. The feed and drinking water were providedadlibitum.

2.2 Methods

2.2.1Modeling of cirrhosis and ascites. After one week of adaptive feeding, 10 rats were randomly selected from the 100 male healthy male SD rats as the blank group. The remaining rats were given 35% phenobarbital solution as drinking water, and after one week, normal drinking water was restored, and the rats were injected subcutaneously with 40% CCl4-vegetable oil solution on the back for modeling. From the 2nd week, subcutaneous injection of 40% CC14-vegetable oil solution was performed three times a week, at a dose of 0.2 mL/100 g. The first dose was doubled. From the 14th week, two rats were sacrificed randomly every week from the model rats, and their liver tissues were sampled for sectioning and HE staining until the formation of cirrhosis was observed. Other two rats were sampled randomly for withdrawing of peritoneal fluid. If peritoneal fluid was withdrawn, it meant that the ascitic cirrhotic model was successfully established[10].

2.2.2Animal grouping. The rat models were randomly divided into five groups: hydrochlorothiazide group (positive group) (15 rats), high-dose ethanol extract group (15 rats), medium-dose ethanol extract group (15 rats), low-dose ethanol extract group (15 rats) and model group (30 rats).

2.2.3Administration method. After modeling, the rats in the blank group began to be administered intragastrically with saline, once a day, and the volume was the same as that of each ethanol extract administration group; the rats in the positive control group began to be administered intragastrically with hydrochlorothiazide at a dose of 8.82 mg/kg, once a day; the rats in the high-dose ethanol extract group began to be administered intragastrically with ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv at a dose of 100 mg/kg, once a day; the rats in the medium-dose ethanol extract group began to be administered intragastrically with ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv at a dose of 50 mg/kg, once a day; the rats in the low-dose ethanol extract group were administered intragastrically with ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv at a dose of 25 mg/kg, once a day; and the rats in the model group were given with saline by gavage, once a day, and the volume was the same as that of each ethanol extract administration group. All the rats above were administered intragastrically at a dose of 1 mL/100 g.

2.2.4Molding standards. In the rats of the model group, obvious abdominal bulge was observed, accompanied by subcutaneous fluctuations;the amount of fluid in the abdominal cavity increased, and the difference was statistically significant; regenerative nodules were observed with naked eye in the livers; and the fibrous septa in the portal area-portal area or portal area-central vein expanded and connected to each other, and the liver lobules were divided to form pseudolobules[11].

2.2.5Sample collection. After the experiment ended, all the rats were fasted for 18 h, and then anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.4 mL/100 g. Subsequently, the abdominal cavity of each of the rats was opened, and the peritoneal fluid was measured, and the blood of the abdominal aorta was collected.

2.2.6Statistical analysis. Statistical analysis of experimental data was performed using SPSS 21.0 statistical software. The measurement data were expressed as mean±SD. One-way ANOVA was used for normal data. For comparisons among groups,LSD-ttest was adopted when variances were homogeneous, and Games-Howell test was adopted when variances were unhomogeneous. The significance level was set at α=0.05.

2.2.7Items observed. The activities, mental status, weight change, hair condition, eating and death of the rats were observed.

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2.2.8Detected indicators and detection methods. (i) Amount of peritoneal fluid. After the last administration, each of the rats was anesthetized and cut 3.0 cm along the midline of the abdomen. Subsequently, a weighed cotton ball was quickly stuffed into the abdominal cavity. After 3 min, the cotton ball that had fully absorbed the peritoneal fluid was taken out and weighed. The difference between the two weights was calculated as the amount of the peritoneal fluid. (ii) Changes in liver functions. A certain volume (3 mL) of blood was collected from the abdominal aorta of each of the rats, let stand at room temperature for 60 min and centrifuged at 3 000 rpm/min for 15 min at 4 ℃, and the serum was transferred into a new centrifuge tube, sealed, and frozen at -20 ℃ for testing. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) were determined by a fully automatic biochemical analyzer. (iii) Concentration of K+in serum. A certain volume (2 mL) of blood was collected from the abdominal aorta of each of the rats, placed in vacuum blood collection tube (non-anticoagulation tube), let stand at room temperature for 60 min and centrifuged at 3 000 rpm/min for 15 min at 4 ℃, and the serum was transferred into a new centrifuge tube, sealed, and frozen at -20 ℃ for testing. The concentration of K+in the serum was detected by automatic biochemical analyzer. (iv) Concentration of aldosterone (Ald) in plasma. A certain volume (3 mL) of blood was collected from the abdominal aorta of each of the rats, placed in EDTA vacuum blood collection tube, let stand at room temperature for 60 min and centrifuged at 3 000 rpm/min for 15 min at 4 ℃, and the serum was transferred into a new centrifuge tube, sealed, and frozen at -20 ℃ for testing. An appropriate amount of plasma was used for measurement, and the operations were performed according to the instructions of the Elisa kits.

3 Results and analysis

3.1 General situation of rats

3.1.1Blank group. The rats were in good mental condition and ate normally. Their body weight increased, hair was orderly and shiny, and activity was normal. No other abnormalities were seen.

3.1.2Model group. The mental state of the rats was not good, showing a certain degree of malaise. They were more manic and irritable. Their feed intake and body gain were lower (compared with those in the other groups); their coat was dull and disorderly; their stool excretion was abnormal, thin and stinky; their activity reduced; and their eyes were dull.

3.1.3Administration groups. The rats in the administration groups had better mental status (compared with the rats in the model group), normal feed intake, shiny coat and better movement. The others were better than the model group.

3.2 Effect of ethanol extract fromL.alata(D. Don) Sch. Bip. ex Oliv on ascites in rats with liver cirrhosisAt the end of the experiment, the rats in the blank group did not show ascites, and the rats in the other groups showed varying degrees of ascites. The model group showed the most peritoneal fluid, followed by positive group, and the high-dose ethanol extract group showed the least peritoneal fluid. The peritoneal fluid in the rats of the high, medium and low-dose ethanol extract groups was less, and as the dose increased, the amount of peritoneal fluid reduced. The amount of peritoneal fluid in the rats of the model group was higher than those of the other groups (P<0.05) (Table 1).

Table 1 Comparison of amount of peritoneal fluid among the groups (Mean±SD,n=8)

GroupnAmount of peritoneal fluid∥gBlank80.031±0.006Model81.068±0.517##Low dose 80.398±0.427Medium dose80.286±0.036∗∗High dose80.225±0.068∗∗Positive8 0.447±0.121#∗∗

Note: Compared with the blank group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

3.3 Effect of ethanol extract from L. alata (D. Don) Sch. Bip. ex Oliv on liver functions in cirrhotic rats with ascites

3.3.1Effect on serum ALT level. Compared with the blank group, the levels of ALT in the other groups increased to varying degrees. The increase in the model group was largest, followed by that in the low-dose ethanol extract groups, and the increase in the high-dose ethanol extract was smallest. There were significant differences between the model group, positive group and low, medium and high-dose ethanol extract groups and the blank group (P<0.05). Compared with the model group, the levels of ALT in the ethanol extract administration groups declined to varying degrees. There was a significant difference between the high-dose ethanol extract group and the model group (P<0.05). The ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv caused ALT decrease in the ascetic cirrhotic rats, and this effect was dose-dependent (more obvious at high dose) (Table 2).

Table 2 Effects of ethanol extract ofLaggeraalata(D. Don) Sch. Bip. ex Oliv on liver functions of rats (Mean±SD,n=8)

GroupSerum ALT∥U/LSerum AST∥U/LSerum ALB∥g/LK+∥mmol/LPlasma Ald∥mmol/LBlank43.52±10.81303.58±57.7931.08±4.726.29±0.82829.86±40.48Model429.71±52.73##893.28±93.28##18.96±2.26##5.87±1.13851.52±48.34Low dose408.72±60.25##855.24±76.81##19.05±2.18##5.75±0.54845.37±42.05Medium dose378.15±54.23##799.82±56.49##19.29±2.95##5.88±0.43804.21±39. 28High dose253.19±46.21##∗505.56±45.98∗17.92±2.31##5.95±0.62 758.06±40.92#∗∗Positive321.04±36.84##692.58±60.35#21.78±1.94# 5.12±0.49# 744.69±50.01#∗∗

Note: Compared with the blank group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

3.3.2Effect on serum AST level. Compared with the blank group, the AST levels of other groups increased to varying degrees. The increase in the model group was the largest, followed by that in the low-dose ethanol extract group. Significant differences were found between the model group, positive group and medium and low-dose ethanol extract groups and the blank groups (P<0.05), while there was no significant difference between the high-dose ethanol extract group and the blank group (P>0.05). Compared with the model group, the levels of AST int the ethanol extract administration groups decreased to varying degrees, and the difference between the high-dose ethanol extract group and the model group was significant (P<0.05). Different doses of ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv all caused decrease in serum AST level, and the effect of high-dose group was more obvious. The results are shown in Table 2.

3.3.3Effect on serum ALB level. Compared with the blank group, the levels of ALT in the other groups decreased to varying degrees. The decrease in the high-dose ethanol extract group was the largest, followed by the model group. There were significant differences between the model group, positive group and low, medium and high-dose ethanol extract groups and the blank group (P<0.01), while no significant difference was found between the high, medium and low-dose ethanol extract groups and the model group (P>0.05). The ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv failed to cause ALB elevation in the serum of cirrhotic rats with ascites. The results are shown in Table 2.

3.3.4Effect on K+concentration in serum. Compared with the blank group, the serum K+concentrations in the other groups decreased in varying degrees; and the decrease in the positive group was significant (P<0.05). There was a positive correlation between the dose of ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv and serum K+concentration. The results are shown in Table 2.

3.3.5Effect on Ald level in plasma. The plasma Ald levels in the high-dose ethanol extract group and positive group were lower than that in the blank group (P<0.05), and there were no significant differences in plasma Ald level between the medium and low-dose ethanol extract group and the blank group (P>0.05). The plasma Ald levels in the high-dose ethanol extract group and positive group were lower than that in the model group (P<0.01), and no significant difference was found in plasma Ald level between the medium and low-dose ethanol extract groups and the model group (P>0.05). The results are shown in Table 2.

4 Discussion

4.1 Stability of rat models of cirrhosis and ascitesAfter a period, the preparation of rat model of cirrhosis and ascites was grasped to a certain extent. After induction by using 35% phenobarbital solution for one week, subcutaneous injection of 40% CCl4-olive oil solution was performed three times a week starting from the 2nd week. Each dose was 0.2 mL/100 g, and the first dose was doubled. From the 14th week on, two model rats were randomly sacrificed at the end of every week and their liver tissues were sampled for sectioning and HE staining until the formation of cirrhosis. Then, two model rats were randomly selected for withdrawing of peritoneal fluid. If peritoneal fluid was withdrawn, it meant that the ascitic cirrhotic rat model was successfully established. The liver tissues were fixed, sectioned and stained by HE. Under the microscope, it could be seen that the normal structure of the liver was obviously damaged; fibrosis was obvious; swelling of hepatocytes was obvious; sinusoids became smaller and even disappeared; a large number of Kupffer cells could be seen in the sinusoids; fatty degeneration was extensive; and a large number of deformed hepatocytes were in the form of strands, extending from the portal vein into hepatic lobules, accompanied by punctate necrosis. It indicates that the rat model of cirrhosis and ascites was successful and stable[12].

4.2 Expressions of ALT and AST in cirrhotic rats with ascitesALT and AST are the most sensitive indicators of hepatocyte membrane damage. The damage of hepatocyte membrane will cause a large amount of ALT and AST to overflow into the blood, resulting in a sharp elevation of ALT and AST in serum. The level of ALT and AST activity can sensitively reflect the degree of damage to hepatocytes.

4.3 Expression of ALB in cirrhotic rats with ascitesSerum ALB is a functional protein specifically secreted by hepatocytes, and it reflects the impairment of hepatocellular functions. Content of ALB decreases when hepatocytes are damaged. In liver disease, decrease in ALB level is usually accompanied by an increase in GLO. Decreased ALB-GLO ratio reflects changes in liver functions[13]. When hypoproteinemia occurs, the colloid osmotic pressure is reduced, which is related to the formation of ascites. Ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv cannot increase serum ALB level. Single Chinese medicine treatment is difficult to achieve good results, so, it should integrate Chinese and Western medicine according to the actual situation, and supplement appropriate amount of ALB to improve the patients’ condition.

4.4 Role of K+in serumMost of K+are present in the cell, and a small amount are in the extracellular fluid. The concentration of K+is relatively constant. The determination of serum K+concentration is actually the determination of K+concentration in extracellular fluid. However, K+in the body often exchange with each other between cells and body fluids to maintain dynamic balance. Therefore, the level of serum K+concentration, to some extent, can also indirectly reflect the level of K+in cells. Severe vomiting or diarrhea, gastrointestinal decompression and extensive use of potassium excretion diuretics and adrenal corticosteroids will cause adrenocortical hyperfunction and hyperaldosteronism. In this experiment, the ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv has a potassium-preserving effect in the treatment of cirrhosis and ascites; there were no significant differences between the high, medium and low-dose ethanol extract groups and the blank group (P>0.05); and hydrochlorothiazide tablets cause hypokalemia in the treatment of cirrhosis and ascites, and the serum K+level in the positive group showed significant difference with that of the blank group (P<0.05).

5 Conclusions

High and medium-dose ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv and hydrochlorothiazide tablets have the effect of resolving ascites. The ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv can reduce the levels of ALT and AST and has a protective effect on the liver. The administration of ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv did not cause hypokalemia, but the administration of hydrochlorothiazide tablets caused hypokalemia. The administration of high-dose ethanol extract ofL.alata(D. Don) Sch. Bip. ex Oliv and hydrochlorothiazide tablets reduced the serum Ald level in cirrhotic rats with ascites.