Effect of Elephantopus scaber L. Extract on Acute Pleurisy in Rats

Yan ZHANG, Jianting GAO, Xinchao SHI, Hailong LIU*, Xin HE

1. Institute of Animal Husbandry and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, China 2. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China

Abstract [Objectives] To study the anti-inflammatory effect of Elephantopus scaber L. extract on acute pleurisy induced by carrageenan in rats, and to explore its anti-inflammatory mechanism. [Methods] The active sites of E. scaber L. were extracted by ethanol reflux method. The extracts of different concentrations of E. scaber L. were used as the study object, and dexamethasone was used as the positive control drug. The anti-inflammatory effects of E. scaber L. extracts were studied by measuring the levels of malondialdehyde (MDA), prostaglandin E2 (PGE2), tumor necrosis factor (TNF-α), interleukin-1β(IL-1β) in pleural fluid and serum nitric oxide (NO), MDA, PEG2, TNF-α, IL-1β in rats with acute pleurisy induced by carrageenan. [Results] E. scaber L. extracts in three doses could reduce the levels of inflammatory factors in pleural fluid and serum, and inhibit acute pleurisy in rats. It was speculated that the anti-inflammatory mechanism was related to the inhibition of the release of inflammatory factors and the antioxidant effect of extracts of three doses of E. scaber L. [Conclusions] This experiment provides a basis for the development and application of E. scaber L.

Key words Elephantopus scaber L., Extract, Acute pleurisy, Anti-inflammatory

1 Introduction

ElephantopusscaberL. is a medicinal plant in the family of Compositae, mainly distributed in Guangdong, Zhejiang and Fujian, China. Its whole plant can be used as medicine[1]. It has the effects of clearing away heat and toxic materials, reducing swelling and diuresis, and is often used for the treatment of cold, acute tonsillitis, conjunctivitis, edema and other diseases[2]. Inflammation is a common comprehensive pathological process in humans and animals. It is a defense-oriented pathological reaction of living tissues with vascular system to inflammatory factors, and it can be divided into acute inflammation and chronic inflammation. Acute inflammation refers to the response of the body in a short time after being stimulated by inflammatory factors. Pleurisy is one of the most common inflammatory diseases, and many life-threatening diseases, such as tuberculosis and pneumonia, are related to pleurisy[3]. Rat pleurisy model induced by carrageenan is an ideal acute inflammatory model, which is often used to test the anti-inflammatory effect of drugs[4]. For rodents, carrageenan injected into thoracic cavity can activate kinase, promote the release of inflammatory factors, activate inflammatory cells (mainly neutrophils) to gather in inflammatory sites. Therefore, with the help of this model, the anti-inflammatory effect of drugs can be studied by measuring the level of inflammatory factors and MDA content.

In this study, the active substances ofE.scaberL. were extracted by ethanol reflux method, and the effects of different concentrations of extracts on acute pleurisy in rats were investigated, and its anti-inflammatory mechanism was preliminarily discussed, which provided a basis for its research and development and application.

2 Materials and methods

2.1 Materials

2.1.1Test animals and plants. Healthy Kunming male rats, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., weighed (250±20.0) g, and the production license number was SCXK (Beijing) 2019-0010.

The purchased rats were fed adaptively for one week, during which it was kept ventilated, and they were forced to fast for 12 h but could drink water before the start of the experiment.E.scaberL. was collected in Dongshan Town, Haikou City, Hainan Province in March 2022.

2.1.2Pharmaceuticals and instruments. (i) Pharmaceuticals. Dexamethasone Sodium Phosphate Injection (5 mg/mL) (Shanxi Zhaoyi Co., Ltd.); Carrageenan (Shanghai Macklin Biochemical Technology Co., Ltd.); Heparin Sodium Injection (Tianjin Biochemical Pharmaceutical Co., Ltd.); Anhydrous Ethanol (Tianjin Huihang Chemical Technology Co., Ltd.); 0.9% Normal Saline Injection (Shiyao Yinhu Pharmaceutical Co., Ltd.); Tween-80 and other solvents (Fuchen Chemical Reagent Co., Ltd.); Rat Nitric Oxide (NO) ELISA Detection Kit (Shanghai Yiyan Biotechnology Co., Ltd.); Elisa Kit for Detecting Malondialdehyde (MDA), Prostaglandin E2(PGE2), Tumor Necrosis Factor (TNF-α) and Interleukin-1β (IL-1β) in Rats (Shanghai Zhenke Biotechnology Co., Ltd.).

(ii) Instruments. Extracting and Concentracting Tank (Beijing Jinfu Renhao Technology Development Co., Ltd.); RE-5205 Rotary Evaporator (Shanghai Yarong Biochemical Instrument Factory); SHD-III Circulating Water Multi-purpose Vacuum Pump (Sunshine Science and Education Instrument Factory, Baoding High-tech Zone); TGL-16B Centrifuge (Shanghai Anting Scientific Instrument Factory).

2.2 Methods

2.2.1Preparation of ethanol extract fromE.scaberL. The effective components ofE.scaberL. were extracted by ethanol reflux extraction. 2 600 g ofE.scaberL. was weighed, refluxed and extracted with 95% ethanol for 2 times. 1：15 ethanol was added for extraction for 2 h at the first time and 1: 10 ethanol for extraction for 1 h at the second time; the primary extracts were combined, concentrated under reduced pressure, and ethanol was recovered. After absorption occurred in the concentration bottle, a proper amount of total ethanol extract was taken. The rest of the liquid medicine was concentrated to extract, 500 mL of hot water was added, and stirred well for later use.

2.2.2Animal model establishment and administration. The experimental animals were randomly divided into 5 groups. The control group was divided into negative control group (5% Tween-80) and positive control group (0.5 mg/kg dexamethasone); the experimental group was divided into low dose group (7.5 g/kg), medium dose group (15 g/kg) and high dose group (30 g/kg). The rats were given intragastric administration (1 mL/hg) once a day for 7 d; 30 min after the last administration, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.4 mL/hg[5], fixed in supine position, shaved at the right thoracic cavity, and disinfected by alcohol wiping; according to the dose of 0.2 mL/hg[6], 1% sterile carrageenan solution was injected into the right pleural cavity[7]between the 7thand 8thribs under the right upper limb to cause inflammation[8-9]. In order to avoid lung injury, this operation should be completed in a short time[10].

2.2.3Sampling and determination. After 4 h of inflammation, the rats were anesthetized, and blood was collected from orbital sinuses before killing. The whole blood was centrifuged at 5 000 r/min for 5 min, and the content of inflammatory factors NO, MDA, PGE2, TNF-α and IL-1β in the supernatant was determined according to the instructions of the kit.

The rats were fixed in supine position, wiped and disinfected with alcohol, and then the skin of chest and abdomen was cut with surgical scissors to expose the muscle layer. 2 mL of saline pleural lotion was injected (including 5 U/mL heparin) into the thoracic cavity[11-12], and it was shaken gently to mix the fluid in the thoracic cavity evenly; the pleural exudate was centrifuged at 5 000 r/min for 5 min, and the supernatant was taken. The content of MDA, PGE2, TNF-α and IL-1β was determined according to the instructions of the kit.

2.2.4Statistics and analysis. SPSS 19.0 was used for statistical analysis. The experimental data were expressed by (mean±standard deviation).T-test and one-way ANOVA were used.P<0.05 indicated that the difference was statistically significant.

3 Results and analysis

3.1 Extract content ofE.scaberL.The results showed that the extraction rate of ethanol extract ofE.scaberL. was 30.58%, and 1 g of the extract was equivalent to 3.27 g of the original drug. In this study, the solvent of the extract was selected as the highest dose, and the medium and low dose solvents were diluted accordingly.

3.2 Content of inflammatory factors in serumThe content of inflammatory factors in serum is shown in Table 1. Compared with the negative control group, the content of serum NO, MDA, PGE2, TNF-α and IL-1β in the positive control group was significantly decreased (P<0.05 orP<0.01);E.scaberL. ethanol extracts in different doses of groups could reduce the content of serum inflammatory factors in different levels. Compared with the negative control group, the high dose, medium dose and low dose groups could significantly reduce the serum TNF-α content (P<0.05 orP<0.01), but there was no significant difference between the high dose group and the positive control group; the content of serum NO, MDA and IL-1β in high and medium dose groups was significantly decreased (P<0.05 orP<0.01), and the effects of high dose group on MDA, TNF-α and IL-1β were significantly better than those in medium dose group; only the content of PGE2in serum was significantly reduced in the high dose group.

Table 1 Content of inflammatory factors in serum of rats n=6)

3.3 Content of inflammatory factors in pleural fluidThe results of inflammatory factors in pleural fluid are shown in Table 2. Compared with the negative control group, the content of MDA, PGE2, TNF-α and IL-1β in pleural fluid in the positive control group was significantly decreased (P<0.05 orP<0.01); in the three dose groups of ethanol extract ofE.scaberL., the content of TNF-α in pleural fluid was significantly decreased; the content of MDA, PGE2and IL-1β in pleural fluid was significantly decreased (P<0.05 orP<0.01) in medium dose group and high dose group, and the effect of high dose group on PGE2was significantly higher than that of medium dose group, but not significantly different from that of positive control group.

Table 2 Content of inflammatory factors in pleural fluid of rats n=6)

4 Discussion and conclusions

Acute pleurisy induced by carrageenan in rats is widely used in screening and evaluating anti-inflammatory drugs, and it is an ideal model of acute exudative inflammation[13]. Inflammatory stimulation can cause the secretion of a large number of inflammatory mediators, and it is easy to detect the level of factors related to inflammatory reaction[14-16]. Therefore, the content of inflammatory mediators in rat pleurisy can be used as an important index to measure inflammation.

NO can regulate inflammatory reaction. When inflammation occurs, stimulants promote the synthesis and release of NO, and then promote the inflammatory reaction; MDA can reflect the reaction between free radicals and lipids; PGE2plays an important role in inflammatory reaction, and can promote the role of other factors and make white blood cells gather in inflammatory areas, thus causing inflammatory reactions such as tissue edema and congestion[17]; TNF-α, as a pro-inflammatory factor, can induce or activate a variety of inflammatory mediators in vivo through a variety of biological pathways, and inflammatory mediators work together to cause tissue damage and apoptosis[18-19]; IL-1β, as one of the most important cytokines involved in inflammatory reaction, can increase the secretion of PGE2, participate in mediating acute inflammatory reaction, and promote leukocyte aggregation in local inflammatory sites.

In this experiment, the ethanol extract ofE.scaberL. could decrease the content of NO, PGE2, TNF-α, IL-1β and MDA in serum and pleural fluid, indicating that the mechanism of the ethanol extract ofE.scaberL. inhibiting acute pleurisy in rats may be related to its anti-oxidation and inhibition of the release of inflammatory factors.

Wanetal.[20]found that high concentration ofE.scaberL. ethanol extract can be used as a hepatoprotective drug for liver injury induced by ethanol, and it is safe and has no toxic and side effects in rats. In order to explore the anti-inflammatory effect ofE.scaberL. water extract, Wen Xianminetal.[21]established a rat toe swelling model induced by carrageenan, and compared the swelling degree of the right hind toe and inhibition rate at different time after administration of carrageenan in each group. The results showed that the toe swelling was inhibited when the dose was 0.45 g/kg (approximately equivalent to 38.67 g/kg crude drug). In order to test the median lethal dose (LD50) of different solvent extracts ofE.scaberL., the acute toxicity test of different solvent extracts ofE.scaberL. was carried out in rats. The results showed that at the dose of 5 000 mg/kg (approximately equivalent to 16.35 g/kg crude drug), the rats did not die[22]. The inflammatory model rats were givenE.scaberL. extract at a dose of 0.4 g/kg (approximately equivalent to 8.25 g/kg crude drug). It was found that compared with the control group, the administration group had obvious inhibitory effect on carrageenan-induced inflammation[23]. The extract used in this experiment was sticky, and the high dose group used the extract solution. In order to ensure the flowing state of the liquid medicine, the final dose groups divided into low dose group (7.5 g/kg according to the crude drug quantity), medium dose group (15 g/kg according to the crude drug quantity) and high dose group (30 g/kg according to the crude drug quantity).

In this experiment, the results showed that the ethanol extract ofE.scaberL. could inhibit the content of NO, MDA, PGE2, TNF-α and IL-1β in pleural fluid and serum of rats with acute pleurisy induced by carrageenan. The anti-inflammatory effect was in the order of high dose >medium dose >low dose, suggesting that its anti-inflammatory mechanism might be related to its inhibition of the release of inflammatory factors and its antioxidant effect.