| The Ophiopogon japonicus is the dry root of Liliaceae ophiopogon plant (Ophiopogon japonicus (Thunb.) Ker-Gawl.), nourishing yin is commonly used in traditional Chinese medicine, it’s sweet, slightly bitter, slightly cold, tropisms the heart, lungs, stomach meridian, nourishing the yin, pure heart and moistening Lungs. Its chemical ingredients are steroidal saponins, high isoflavones, polysaccharides, essential oils and amino acids. Modern pharmacological studies have shown that Maidong has the effect of protecting the cardiovascular system, anti-aging, anti-hypoxia, enhance immunity, hypoglycemic. In recent years, a growing number of studies have found the extract has anti-inflammatory effects, which can play a significant inhibition in neutrophil respiratory burst cell activation and expression of chemotactie factors and other channels.Inflammation refers to a living tissue of vascular system on the inflammatory factors in a defense dominated by various kinds of cells, various factors involved in the complex defense system. Inflammation is one of the most common clinical pathological process, the main manifestations of inflammation are local tissue deterioration, liquid and cell exudation, proliferation etc. The development of inflammation is closely related to many serious diseases, including acute inflammatory diseases such as sepsis, systemic inflammatory response syndrome, chronic inflammatory diseases such as diabetes, atherosclerosis, cardiovascular disease and Alzheimer’s disease and so on. Endotoxin is a kind of sugar and lipid complex release in the growth process or after death cleaved the cell wall of gram-negative bacteria, in many factors that can lead to inflammation, endotoxin is the most common and serious pro inflammatory cytokines. Endotoxin enter into the body can induce internal monocyte-macrophage cells release large amounts of inflammatory mediators, such as NO, TNF-a and so have a very serious inflammatory injury, can cause tissue damage and promote the inflammatory cascade, induced the new inflammation medium or cytokine, which mediates the development of a variety inflammation.This experiment is to explore the anti-inflammatory effects of Maidong, first we obtained the water extract and alcohol extract, then extract dichloromethane, n-butanol, and water extraction site step by step, adopt inflammatory reaction model of NO generation and TNF-a expression in LPS-induced RAW264.7 cells to inspect, finally compound were separated and identified to provide theoretical basis for clinical application and the material basis of anti-inflammatory activity efficacy. This paper mainly expounds from following aspects:After obtaining the extraction of Radix Ophiopogonis by water and alcohol solvent, the Ophiopogon japonicus extract treated RAW264.7 macrophages were investigated, found that the water extract (202.6μg/ml) and alcohol extract (184.3μg/ml) of Ophiopogon japonicus at low concentration promote macrophage growth proliferation, enhanced cell viability. But, it had an inhibitory effect on the growth and proliferation of cells at high concentration.LPS induced RAW264.7 cells can secrete a large number of NO and TNF-a, after treatment of water extracts and alcohol extracts from Radix Ophiopogonis can significantly inhibit the macrophage to release NO and TNF-a (P<0.01). The inhibitory effect of the middle dose (50μg/ml) and high dose (100μg/ml) of the two extracts on NO was better than that of the positive drug dexamethasone. In addition, there was no significant difference in the inhibition of NO secretion between the two extracts. Water extract of middle dose (50μg/ml) and high dose (100μ,g/ml) for the inhibitory effect on TNF-a was slightly better than positive drug dexamethasone, although alcohol extract had inhibitory effect, but the effect was worse than water extract and positive drug.The water extract of Ophiopogon japonicus was extracted for the dichloromethane, n-butanol and water fraction and acted on RAW264.7 macrophage. It found that water extraction (166.5μg/ml), n-butanol extracts (357.4μg/ml) and dichloromethane extracts (422.4μg/ml) of Radix Ophiopogonis can promote macrophage proliferation and enhance the vitality of cells at low concentration, where have an inhibitory effect on the growth and proliferation of cells at high concentration.After treatment of LPS induced RAW264.7 macrophages by different Ophiopogon japonicus extract. The amount ofNO and TNF-in the supernatant of cell culture medium was significantly decreased (P<0.05). The water part had the best inhibitory effect on NO, followed by n-butanol and dichloromethane fraction. The low dose (25 g/ml), middle dose (50 g/ml) and high dose (100 g/ml) of the water part were superior to the dexamethasone group. For the inhibition effect on TNF-a, the n-butanol fraction had the best effect, followed by dichloromethane fraction, and water fraction. The middle dose (100 g/ml) and high dose (200 g/ml) of n-butanol fraction have better effect than the positive drug. The effect of high dose of dichforomethane (200 g/mt) was similar to the positive drug.After extraction of Radix Ophiopogonis by heating reflux method, using column chromatography, high performance of preparative liquid chromatography and recrystallization etc. technical means to separate and purify chemical composition of Radix Ophiopogonis. Then 5 compounds and 2 groups of isomers was obtained, which has four flavonoids, namely methyl Ophiopogon flavanone A and methyl Ophiopogon flavanone B, methyl Ophiopogon dihydro flavonoids A and methyl Ophiopogon dihydro flavonoids B, two steroidal saponins compounds, three fatty acids compounds.In summary, this study successfully established LPS induced RAW264.7 cells secrete inflammatory cytokines in vitro models of inflammatory response, verify the regulation effect of Ophiopogon japonicus extracts on inflammatory reaction in vitro, and Radix Ophiopogonis containing compounds were isolated and identified to research the regulatory effect of compound monomer on the inflammatory response. Therefore the paper provide new research directions and theoretical basis for clinical treatment of inflammatory diseases. |
PDF Full Text Request