Anti-Inflammatory Effect Of Supercritical-Carbon Dioxide Fluid Extract From Flowers And Buds Of Chrysanthemum Indicum Linnen

ObjectiveChrysanthemum indicum Linne.(C. indicum) is a traditional Chinese medicine (TCM), which has been used to treat various inflammation-related diseases with high efficacy and low toxicity for several centuries. Currently, the supercritical-carbon dioxide fluid extraction (SCFE) technology has been successfully applied to the extraction process of C. indicum. The SCFE extract of C. indicum (CFE) has been widely used as a fine material in many TCM preparations, functional foods, cosmetics, and toiletries. Most importantly, the CFE is a main ingredient of a TCM recipe named Compound C. indicum Soft Capsule (also known as CPZ in previous studies), an anti-influenza product which activity closely relating to its anti-inflammatory features. However, the chemical composition of CFE whether it has anti-inflammatory activity are unknown so far. Thus, in this study, we analyzed the chemical ingredients of CFE by Gas Chromatography-Mass Spectrometry (GC-MS) analysis and High Performance Liquid Chromatography-Photodiode Array Detector (HPLC-PAD) analysis, and examined the anti-inflammatory activity on various animal models.Methods1The technology investigation and phytochemical study of CFEThis paper optimized supercritical carbon dioxide extraction technology of wild chrysanthemum flower by adopting the orthogonal design method. We experimented on L9(34) orthogonal test table by selecting extraction pressure, extraction temperature, extraction time as factors, and extraction rate as index with three levels.This paper extracted the CFE with n-hexane and70%methanol respectively. And we analyzed the chemical ingredients of CFE by Gas Chromatography-Mass Spectrometry (GC-MS) analysis and High Performance Liquid Chromatography-Photodiode Array Detector (HPLC-PAD) analysis.2The anti-inflammatory effect of CFEKM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration, the xylene-induced mouse ear edema model was duplicated, the weight difference of ears was measured and the degree of ear edema and the inhibition rate were evaluated.KM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration, the acetic-acid-induced vascular permeability enhancement model was duplicated, the OD difference of peritoneal lavage fluids was measured and the degree of vascular permeability enhancement and the inhibition rate were evaluated.KM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration, the carrageenan-induced mouse paw edema model was duplicated, the volume difference of mouse paw was measured and evaluating the degree of edema and the inhibition rate were evaluated.KM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration, the carrageenan-induced mouse paw edema model was duplicated.4h after the carrageenan challenge, mouse was sacrificed. The paw was collected for the analysis of MPO, MDA, TNF-α, IL-1β, IL-6, NO, PGE2, iNOS and COX-2as well as the histopathological evaluation. The liver tissue was collected for the analysis of SOD, GPx and GRd.KM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration, the cotton pellet-induced rat granuloma formation model was duplicated. Weighed the granuloma and compared differences between groups.3The effect of CFE against LPS-induced acute lung injuryKM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration. The LPS-induced acute lung injury model was duplicated. Mice of all groups were monitored and the time when any animal died was recorded every6hours up to120hours. Then the mortality rate of each group within120hours was calculated and compared using the Kaplan Meier methods. KM mice was with pretreatment with CFE (40,80and120mg/kg, p.o.) for7days (q.d.).1h after the last administration. The LPS-induced acute lung injury model was duplicated. The bronchoalveolar lavage fluid (BALF) was prepared for protein contents and cell counts analysis. The lung tissue was collected for the analysis of SOD, GPx, CAT, MPO, MDA, TNF-a, IL-1β, IL-6as well as the histopathological evaluation. The lung tissue was also collected for W/D ratio measure as well as for the western blot assay of TLR4, MyD88and NF-κB.4. Preliminary study of acute toxicity of CFEThe acute toxicity study was performed via combining median lethal (LD50) experiments and maximum tolerance dose (MTD) tests for a single dose toxicity study.Results1The technology investigation and phytochemical study of CFEFactors influencing the extraction rate of progression is extraction pressure> extraction temperature> extraction time. The best optimized supercritical carbon dioxide extraction technology for wild chrysanthemum flower was as follows:25MPa extraction pressure,45℃extraction temperature for3h. The extraction yield was5.12%.This paper identified35kinds of ingredients in the CFE by GC-MS, the main components were as follows:eucalyptol (3.091%), a-thujone (2.186%), β-thujone (2.169%), d-camphor(8.582%), cis-verbenol(4.720%), endo-borneol(7.845%), bornyl acetate(2.948%), thymol(3.071%), β-caryophyllen(3.336%), cis-β-farnesene (2.270%), α-curcumene (5.932%), caryophyllene oxide (8.460%), α-gurjunene (2.161%), aromandendrene (2.280%), α-bisabolol (2.289%), longifolenaldehyde (2.572%), α-bisabolol oxide (2.600%), α-linolenic acid (2.130%), ethyl octadec-9,12-dienoate (2.470%)This paper identified5kinds of ingredients in the CFE by HPLC, they were chlorogenic acid, luteolin-7-glucoside, linarin, luteolin and acacetin.2The anti-inflammatory effect of CFEThe results indicated that CFE significantly attenuated xylene-induced ear edema, decreased acetic acid-induced capillary permeability, reduced carrageenan-induced paw and inhibited the cotton pellet-induced granuloma formation, in a dose-dependent manner. Histopathologically, CFE abated inflammatory response of the edema paw. Preliminary mechanistic studies demonstrated that CFE decreased the MDA level via increasing the activities of anti-oxidant enzymes (SOD, GPx and GRd), attenuated the productions of NF-κB, TNF-a, IL-1β, IL-6, PGE2and NO, and suppressed the activities of iNOS and COX-2.3The effect of CFE against LPS-Induced Acute Lung InjuryData indicated that CFE pretreatment possessed potential preventions against mortality in ALI mice induced by LPS.Results revealed that pretreatment with CFE abated LPS-induced lung histopathologic changes, reduced the wet/dry ratio and pro-inflammatory cytokines productions (TNF-a, IL-1β and IL-6), inhibited inflammatory cells migrations and protein leakages, suppressed the levels of MPO and MDA, and up-regulated the abilities of anti-oxidative enzymes (SOD, CAT and GPx). Furthermore, the pretreatment with CFE down-regulated the activations of NF-κB and the expressions of TLR4/MyD88.4Preliminary study of acute toxicity of CFEData of preliminary acute toxicity indicated that the LD50was uncountable and the MTD was more than4g/kg for a single dose toxicity study.ConclusionThis paper optimized supercritical carbon dioxide extraction technology of wild chrysanthemum flower by adopting the orthogonal design method. The best optimized technology is25MPa extraction pressure,45℃extraction temperature for3h. The extraction yield was5.12%.This paper firstly analyzed the chemical composition of CFE by GC-MS combining HPLC-PAD. Thirty five compounds were identified by GC-MS, and five compounds with anti-inflammatory activity, chlorogenic acid, luteolin-7-glucoside, linarin, luteolin and acacetin, were reconfirmed and quantified by HPLC-PAD. This study systematically investigated the anti-inflammatory property of CFE. The result provides the first evidence for the anti-inflammatory application of CFE, and suggests which mechanism may relate to anti-oxidation and regulation of inflammatory factors. In addition, the results also demonstrated that CFE can effectively attenuate the LPS-induced acute lung injury in mice. The protective effect of CFE was associated with the down-regulation of TLR4/MyD88/NF-KB pathway. These experimental results suggested that CFE was a potential therapeutic drug for acute lung injury.