Preliminary Experimental Study on Anti-inflammatory Effect of Extract from Excoecaria cochinchinensis Lour. as a National Medicine

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

Abstract [Objectives] To study the anti-inflammatory effects of water extract and ethanol extract from Excoecaria cochinchinensis Lour. [Methods] The acute and chronic inflammation models of mouse paw edema induced by cotton ball granuloma and carrageenan were established respectively, and the anti-inflammatory effects of water and ethanol extract from E. cochinchinensis Lour. on acute and chronic inflammation in mice were evaluated. The experimental mice were equally divided into three groups of high, medium and low dose of E. cochinchinensis Lour. (60, 40 and 20 g/kg), dexamethasone (0.006 g/kg) group and blank group (distilled water). In the cotton ball granuloma test in mice, the mice were given intragastric administration continuously for 7 d after cotton ball implantation. In the experiment of inflammation induced by carrageenan, the mice in each group were given intragastric administration once a day for 7 d, and the inflammatory model was made after the last administration. [Results] The granuloma inhibition rates of mice in the high, medium and low dose groups of water extract and ethanol extract from E. cochinchinensis Lour were significantly lower than those in the blank group (P<0.05). The degree of paw edema in each dose group of water and ethanol extract from E. cochinchinensis Lour. was significantly lower than that in the blank group (P<0.05). [Conclusions] The water and ethanol extracts from E. cochinchinensis Lour. had significant anti-inflammatory effects.

Key words Excoecaria cochinchinensis Lour., Extract, Anti-inflammation, Cotton ball granuloma, Paw edema

1 Introduction

The national medicineExcoecariacochinchinensisLour. comes from the whole plant of genus of Excoecaria in the Euphorbiaceae Juss. family. This plant is first included in theListofYunnanMedicinalPlants, and it is also known as Yebeihong, Jinsuoyu and so on.ChineseMateriaMedicarecords that the medicine has the functions of dispelling rheumatism, dredging channels and collaterals, promoting blood circulation and relieving pain. It is used to treat mumps, tonsillitis, angina pectoris and other diseases[2], and Vietnamese people use it to treat urinary tract pain[3]. As a medicinal and ornamental plant,E.cochinchinensisLour. is cultivated in Guangdong, Guangxi, Yunnan and Taiwan, and there are wild species in Longzhou, Guangxi. There are 6 species and 1 variant of Excoecaria plants in China, while there are 40 species of Excoecaria plants in the world, mainly distributed in the tropical regions of Asia, Africa and Oceania[4]. 6 flavonoid glycosides were isolated fromE.cochinchinensisLour. by Li Ziyan[5]in the study of its chemical constituents. Flavonoid glycosides can relieve pain. Related studies have reported that the plant ofE.cochinchinensisLour., a national medicine, contains gallic acid, ursolic acid, kaempferol and diterpenoids[6-9].

At present, there are no related research reports on the anti-inflammatory experiment ofE.cochinchinensisLour.at home and abroad. As the main producing area and clinical application area ofE.cochinchinensisLour., Guangxi has a unique advantage in the development of medicinal value ofE.cochinchinensisLour. Therefore, in this study, the anti-inflammatory effects of water extract and ethanol extract fromE.cochinchinensisLour. were studied by using modern pharmacology, pharmacodynamics, experimental zoology and other experimental techniques. It is of great significance to finding new anti-inflammatory drugs, developing and making full use of Guangxi medicinal plant resources, and promoting the development of Guangxi ethnic medicine industry.

2 Materials and methods

2.1 Materials

2.1.1Experimental animals. Kunming mice, SPF class, male, weighing 18-22 g, were provided by the Experimental Animal Center of Guangxi Medical University, license No.SCXK Gui 2009-0002.

2.1.2Raw materials and reagents. The raw material produced in Fuchuan County, Hezhou City, Guangxi was identified as the whole plant ofE.cochinchinensisLour. of Euphorbiaceae by Associate Professor Teng Jianbei from Department of Traditional Chinese Medicine, Guangxi University of Chinese Medicine. Penicillin sodium for injection, batch No.S070631, Shandong Lukang Pharmaceutical Co., Ltd.; streptomycin sulfate for injection, batch No.070202, Shandong Lukang Pharmaceutical Co., Ltd.; dexamethasone acetate, batch No.1203082, Anhui Jintaiyang Biochemical Pharmaceutical Co., Ltd.; carrageenan, batch No.YY13755, Shanghai Yuanye Biotechnology Co., Ltd.; chloral hydrate, batch No.20130513, Chengdu Kelong Chemical Reagent Factory.

2.1.3Instruments. Electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; vertical pressure steam sterilizer, Shanghai Xunbo Industrial Co., Ltd. Medical Equipment Factory.

2.2 Methods

2.2.1Preparation of samples. 500 g of crude powder ofE.cochinchinensisLour. was extracted with 10 L of distilled water for 3 times. When the extract was combined, filtered and concentrated, three groups of water extracts fromE.cochinchinensisLour. were obtained, respectively, equal to 60, 40, 20 g/kg of crude drug. In addition, 500 g ofE.cochinchinensisLour. powder was extracted with 10 L of 75% ethanol solution for 3 times. When the extract was combined, filtered and concentrated, three groups of ethanol extracts fromE.cochinchinensisLour. were obtained, respectively, equal to 60, 40, 20 g/kg of crude drug. 0.48 g of penicillin sodium for injection and 0.8 g of streptomycin sulfate for injection were mixed and dissolved into 80 mL in distilled water to get double antibody test solution, and it was shaken well and stored at -20 ℃.

2.2.2Cotton ball granuloma test in mice[4]. 96 healthy Kunming mice, male, were weighed. They were randomly divided into 8 groups (12 in each group): blank control group, positive control group (dexamethasone, 0.006 g/kg), high, medium and low dose groups of water extract fromE.cochinchinensisLour. (60, 40, 20 g/kg), and high, medium and low dose groups of ethanol extract fromE.cochinchinensisLour.(60, 40, 20 g/kg). After the animals were anesthetized, two 10 mg of cotton balls which had been sterilized under high pressure and dripped with 0.1 mL of double antibody test solution were implanted into the armpits of mice on both sides. After suturing the incision, iodine was applied to fight infection. The mice in each group were given the corresponding drugs by intragastric administration the next day, and the blank group was given the corresponding volume of distilled water, once a day for 7 d. The mice were killed 12 h after the last administration, and the cotton balls of granulation tissue were stripped off. The wet weight of cotton ball was precisely weighed, baked at 60 ℃ to constant weight, and then precisely weighed, and the granuloma weight was calculated as follows.

2.2.3Experiment of paw edema induced by carrageenan in mice[5]. 96 Kunming mice were weighed, grouped and given at the same dose as in Section2.2.2. Each group was given intragastric administration according to the volume of 20 mL/kg, once a day for 7 d. After the last administration, it was marked with a marker pen at the same height of the hind foot of each mouse, and the normal volume of the toe was measured by paw edema volume meter and recorded. 0.02 mL of 1% carrageenan was injected subcutaneously into the toes of mice. The paw edema volume was measured at 0.5, 1.0, 1.5, 2.5, 3.5 and 4.5 h after inflammation, and the inhibition rate was calculated as follows:

Inhibition rate (%)= (Post-inflammatory V2—Pre-inflammatory V1)/Pre-inflammatory V1×100%.

3 Results and analysis

3.1 Effect on cotton ball granuloma in miceThe granuloma weight of mice in each dose group of water and ethanol extract fromE.cochinchinensisLour. was significantly lower than that in the blank group, and the difference was significant (P<0.05). And the low-dose group of water extract fromE.cochinchinensisLour. had the most significant inhibition effect on the wet weight of cotton ball granuloma in mice, as high as 15.26%. The results are shown in Table 1.

GroupDose∥g/kgWet weight ofgranuloma∥mgWet weight inhibitionrate∥%Dry weight ofgranuloma∥mgDry weight inhibitionrate∥%Blank-0.068 1±0.008 1-0.020 1±0.002 5-Positive control0.0060.047 9±0.006 3∗∗59.610.015 2±0.001 1∗∗23.01High dose of water extract600.062 5±0.007 6∗∗9.740.016 1±0.001 8∗∗15.78Medium dose of water extract400.059 6±0.005 8∗∗15.180.016 8±0.002 1∗∗15.12Low dose of water extract200.058 9±0.008 7∗∗15.260.016 9±0.001 6∗∗13.48High dose of ethanol extract600.061 3±0.008 1∗∗12.430.017 1±0.004 1∗11.42Medium dose of ethanol extract400.059 1±0.008 1∗∗14.070.017 7±0.002 5∗∗10.17Low dose of ethanol extract200.573 0±0.007 2∗∗14.350.017 9±0.002 18.69

Note:*P<0.05,**P<0.01 compared with the blank control group.

3.2 Effect on paw edema induced by carrageenan in miceThe paw edema degree of mice in each dose group of water and ethanol extract fromE.cochinchinensisLour. was significantly lower than that in the blank group, and the difference was significant (P<0.05). And the paw edema inhibition effect of high dose group of water and ethanol extract fromE.cochinchinensisLour. was more obvious than that of medium and low dose groups. The results are shown in Table 2.

4 Conclusions and discussions

Flavonoids have anti-inflammatory, anti-bacterial, anti-oxidant and anti-tumor pharmacological effects. Rheumatoid arthritis (RA) is currently considered to be an autoimmune disease characterized by inflammatory synovitis. Total flavonoids can inhibit the secretion of inflammatory factors such as serum interleukin (IL-1β) and tumor necrosis factor (TNF-α)[11]. In the occurrence of RA, the two factors mostly play a synergistic role, also known as "sister factors"[12].

GroupPre-inflamm-atory V1∥mLPost-inflammatory V2∥mL0.5 h1.0 h1.5 h2.5 h3.5 h4.5 hInhibition rate∥% 0.5 h1.0 h1.5 h2.5 h3.5 h4.5 hBlank group0.251±0.0160.262±0.0210.276±0.380.305±0.0290.311±0.050.275±0.0140.260±0.015------Positive group0.172±0.0230.184±0.0290.192±0.0160.182±0.0270.207±0.0290.239±0.021∗∗0.191±0.019∗5.49 5.529.8512.41-1.527.75High dose of water extract0.198±0.0170.211±0.015∗∗0.204±0.037∗∗0.250±0.021∗∗0.241±0.015∗∗0.240±0.023∗∗0.202±0.014∗∗5.21 1.76 21.05 20.28 17.59 2.38Medium dose of water extract0.214±0.0110.221±0.024∗∗0.261±0.014∗0.263±0.028∗0.254±0.021∗∗0.239±0.016∗∗0.201±0.011∗∗4.31 17.8118.2916.658.37 -6.80Low dose of water extract0.218±0.0140.225±0.016∗∗0.259±0.021∗0.252±0.024∗∗0.237±0.022∗∗0.244±0.013∗∗0.204±0.015∗∗4.52 17.25 15.18 9.31 11.72 -5.62High dose of ethanol extract0.201±0.0190.215±0.017∗∗0.230±0.012∗∗0.251±0.020∗0.248±0.015∗∗0.227±0.011∗∗0.204±0.012∗∗4.81 8.10 9.24 1.61 -4.56-9.84Medium dose of ethanol extract0.203±0.0120.215±0.016∗∗0.210±0.017∗∗0.258±0.0260.250±0.024∗∗0.238±0.0220.205±0.012∗∗4.79 0.71 4.12 1.31 -4.21 -12.51Low dose of ethanol extract0.216±0.0140.221±0.017∗∗0.247±0.013∗∗0.250±0.0180.243±0.017∗∗0.233±0.015∗0.204±0.016∗∗4.47 9.21 1.76 1.08 -0.38 -10.82

Note:*P<0.05,**P<0.01 compared with the blank control group.

Inflammation is a very complex physiological system of the body, and it usually changes the normal physiological indexes of the body and induces a variety of acute and chronic inflammatory diseases[10]. Inflammatory reaction is a complex process in which a variety of mediators participate and inflammatory factors struggle with the body until reaching a balance, while traditional Chinese medicine plays an anti-inflammatory effect through many ways and links. Traditional Chinese medicine has become the preferred object of anti-inflammatory drug research because of its wide variety, complex and diverse components, wide pharmacological effects, low side effects and so on. In recent years, researchers at home and abroad are more and more interested in this field. Through the cotton ball granuloma experiment, we simulated the granulation tissue formation ability in the late stage of inflammation, used carrageenan as an inflammatory agent to cause local acute exudative inflammatory edema in mice, and observed the effects of water and ethanol extract fromE.cochinchinensisLour. on cotton ball granuloma and carrageenan-induced paw edema in mice. The results showed that the high, medium and low doses of water and ethanol extract fromE.cochinchinensisLour. could significantly inhibit the tissue swelling in the early stage of inflammation, and on the whole, the effect of water extract fromE.cochinchinensisLour. was more significant than that of ethanol extract fromE.cochinchinensisLour. However, the specific mechanism of anti-inflammation is not clear, which needs to be further studied and determined. In addition, the traditional method of reflux extraction was used in this experiment, and the components of water and ethanol extract were very complex. The specific effective parts and chemical components need to be further isolated, purified and identified. It is necessary to identify the effective anti-inflammatory components ofE.cochinchinensisLour. extract, so as to provide experimental basis for further discussion of its anti-inflammatory mechanism and the development of its clinical application value.