| Hibiscus manihot is an annual herb of the family Osmanthaceae.It is mainly distributed in the natural area of Hebei Province.It is a special plant for both medicine and food.It is used as a Chinese herbal medicine to treat Wulin,edema,and can be used for external treatment.Studies have shown that Hibiscus manihot contains a variety of active substances,and flavonoids are one of the main active ingredients.At present,the research on the Hibiscus manihot is mainly focused on cultivation.The extraction process and reports on its active substances are mainly concentrated on polysaccharides,crude flavonoids and volatilse oils.Further separation,extraction and identification of active substances and their biological activities Aspect research is relatively scarce.Therefore,in this study,the traditional solvent extraction method was used to extract and separate the total flavonoids from the flower Hibiscus manihot.Then,the macroporous resin method was used to further purify and purify the Hibiscus manihot extract.At the same time,another Hibiscus manihot immersion The pastes were extracted with different solvents,and the dichloromethane extraction phase was selected for separation.The isolated monomer compounds were identified by nuclear magnetic resonance technique;the acute extracts of mice were determined by different concentrations of total extracts and monomer compounds.The effects of different concentrations of monomeric compounds on five kinds of cancer cells were determined by MTT.Mouse peritoneal macrophages(RAW264.7)were induced by lipopolysaccharide,and different concentrations of monomers were determined by ELISA.The anti-inflammatory activity of the compound lays a foundation for the study of related mechanisms.The research results provide a scientific basis for the comprehensive utilization of Hibiscus manihot,and have certain practical guiding significance.The main findings are as follows:(1)The quercetin monomer compound was repeatedly isolated and purified from the total extract of Hibiscus manihot by macroporous resin method and various column chromatography.The yield of flavonoids in different gradient elution phases was determined as follows.The flavonoid content is 19.5%,the total flavonoid content in the aqueous phase is 14%,the total flavonoid content in the 30% alcohol phase is 29.5%,the total flavonoid content in the 50% alcohol phase is 48%,and the total flavonoid content in the 70% alcohol phase is 36%,90%.The total flavonoid content of the alcohol phase was 30.5%.Three monomeric compounds were isolated from the dichloromethane extract phase and the 50% alcohol phase,which were ?-sitosterol,pentahydroxymethylfurfural,and quercetin(2)Different concentrations of Hibiscus manihot extract and three monomeric compounds acted on the mouse acute inflammation model.For the mouse ear swelling model: the high dose group of Hibiscus manihot total extract(1 g/mL),quercetin high Dosage group(5 mg/mL),medium dose group(2.5 mg/mL),low dose group(1.25 mg/mL),high dose ?-sitosterol(5 mg/mL),medium dose group(2.5 mg/)mL)significantly inhibited ear swelling in mice(P<0.01);low dose of ?-sitosterol(1.25 mg/mL)and total extract of Hibiscus manihot(0.33 g/mL)significantly inhibited mouse ears Swelling(P<0.05);5-HMF had no significant difference in inhibiting ear swelling in mice(P>0.05).For the toe swelling model: the high dose group of Hibiscus manihot total extract(1 g/mL),the high dose of quercetin(5 mg/mL),the middle dose group(2.5 mg/mL),and the ?-sitosterol high dose group(5 mg/mL)significantly inhibited mouse toe swelling(P<0.01);quercetin low dose group(1.25 mg/mL),?-sitosterol medium dose group(2.5 mg/mL)and The high dose group of Hibiscus manihot extract(1 g/mL)significantly inhibited the swelling of the toes in mice(P<0.05).The difference of 5-HMF in inhibiting the swelling of the toes in mice was not significant(P>0.05).The pathological section showed that: compared with the normal ear and toe,the model group had hyperemia of the ear and toe vessels,subcutaneous connective tissue and intermuscular edema,and a large amount of neutrophil infiltration,which was characterized by acute suppurative inflammation;Compared with the animal model group,dexamethasone,quercetin each dose group,?-sitosterol high dose group(5 mg/mL),medium dose group(2.5 Mg/mL)inflammatory cell exudation is significantly less,edema is reduced.The experimental results show that quercetin and ?-sitosterol obtained from the Hibiscus manihot have certain anti-inflammatory activity.(3)The inhibition rates of three monomeric compounds,quercetin,5-HMF and ?-sitosterol,against PC-3,BGC-823,MGC-803 and EC-109 were determined by MTT assay.The results showed that quercetin had a better inhibitory effect on PC-3 and BGC-823 cells,but not on MGC-803 and EC-109 cells.The corresponding IC50 were: 16.381±1.468 ??,13.161±1.352 ??;>100 ??;>100 ??.5-HMF has no obvious inhibitory effect on four kinds of tumor cells,and its IC50 is greater than 100;?-sitosterol has better inhibitory effect on PC-3 and MGC-803 cells,but not on BGC-823 and EC-109 cells.The corresponding IC50 are: 16.240.±1.472 ??,52.298±1.26 ??;>100 ??;>100 ??.That is,quercetin and ?-sitosterol have a certain inhibitory effect on tumor cells.(4)The results of enzyme-linked immunosorbent assay(ELISA)showed that ?-sitosterol and quercetin inhibited the release of pro-inflammatory factors TNF-? and IL-1? by LPS-induced Raw264.7 cells.And as the dose increased,the amount of secretion decreased,in a dose-dependent manner.Compared with the LPS group,the concentration of quercetin at 16 ?M reduced the release of TNF-? by 49.34% and the release of IL-1? by 53.02%.The secretion of TNF-? and IL-1? by 64 ?M ?-sitosterol decreased by 29.18% and 41.17%,respectively.5-HMF had no effect on the secretion of the two cytokines,ie no effect.Among them,quercetin has the strongest inhibitory effect on the release of TNF-? and IL-1?. |
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